Relates to the epdmiological conecpt in Joralemons book

Не выводить, если нет значения

Easy way for large corporations to dodge accountability

Major child labor issue

Child labor in a company that claims zero tolerance -- What companies are being accused? Apple, Samsung, Sony

High risk industry -- Raises questions about occupational safety data and how these deaths occurred and were they preventable

DRC Cobalt mines but also what other types of mines

This sentence is important to me because it shows the fear of any parent right through is words of worry. The fear of their daughters being dishonored. In this day and age there are still cases such as where a woman is disrespected and or mistreated just because she is a woman, and this is today where society has changed its views and is less misogynistic. I can only imagine the scenarios that play in Jourdon Anderson mind back then. I believe He is a great father.

So they only care about the financial reasons for not fishing in oyster reefs

SO WHY AREN'T WE DOING ANYTHING ABOUT IT

as they are using the 2010 fmp

yet they don't know if they are overfished or not

at what stage were they sampled? right as the trawl unloads? or when the fish are actually sorted through

so given the uncertainty, why continue to trawl?

underestimate

accounts for less than have of estuarine ecosystems in nc

we know that fish outnumber shrimp in catch abundance according the the 2016 brown study.

misquoting this research, it is just polychaetes that are growing in numbers due to dead bycatch creating a trophic shift.

where is the research for this?

I believe there should be this rule for all school, Elementary, Middle School, and High school because high school students are allowed to use their phones and it makes it a distraction for them in their education

fdfd

This is not measurable.

makes the body paragraphs interesting and informative to keep the reader engaged while sharing facts or other information

keeps the essay organized and focuses on the topic of the essay to keeps the reader engaged

Answering these questions help shape the research papers that you write and it organizes all the information

Why do researcher need to revise or change their research questions instead of sticking to the original one?

yeah agree! research is better when we truly want to do it, not just we have to.

It seems like research is ongoing process instead of one time task.

This makes me realize that all the questions cannot be answer in the same way . That means research is not just finding information but finding the right information.

This is actually a really helpful thing to read right now, because for my group project I'm attempting to detect content on Bluesky that promotes eating disorders/disordered eating habits. It's a good reminder at least for my Dream version of the program I'm creating that only scanning keywords allows for loopholes.

This reminds me of ED twitter a lot, where young women (usually) post their bodies and ask for honest critiques of their body to worsen their own perception of their body. I think its an incredibly harmful, and often overlooked, form of sel-fharm because people see it as just being young and on social media.

The internet certainly accelerates dangerous communities, especially when users are lonely and struggle with mental health. The incel community has only continued to expand and grow, developing into the looksmaxxing and blackpill communities today and even connecting with major right wing content creators and politicians.

This surprised me because it shows how social validation online can become internalized in a strange way. Instead of avoiding negativity, people recreate it themselves, almost to confirm a belief they already have. That makes online harm feel psychological, not just social.

Doomscrolling isn’t a totally new impulse, people have always sought information during scary times, but social media makes it much harder to stop because the stream is endless and constantly updating. That’s why newer platforms can intensify anxiety even when the underlying desire (staying informed) is understandable.

Auvelity

VerB　のタグはなくす

a strange conceit. original design implies that there is some authoritative consensus building strict boundaries of what writers write about and why.

"being left to itself" is too vague. Humans are a part of nature and always will be. There is no separation, therefore it cannot be left to itself. We can identify behaviors that are more or less destructive to individuals and ecosystems thriving, but we can't say nature must just be left to itself.

caused by multiple genes

I find this interesting, as it's basically Carnegie saying that the wealth and power is better served in the hands of a few select people, rather than in the hands of the masses of people. It's interesting, because it's effectively the antithesis of the United States. The U.S. was founded on the idea that common people would have a say in government and in what the government spends money on. Individuals having freedom to do as they please. Carnegie is arguing that by sacrificing the freedom to purchase things as an individual, you enable a singular person to make decisions on what's best for the community. I think it then begs to question as to why we would all want to trust one person to decide what will benefit the common people the most, rather than letting the common people decide.

The way I hear this, is it's Carnegie saying that competition leads to improvements in overall life. Analysing it from a military perspective proves that this is actually true. The Second World War had dozens of nations fighting and competing to out fight the others, with the Allied and Axis powers being full of many major nations. The technology they developed to try and defeat each other would end up leading directly to the invention of the microwave, men in space, nuclear power, jet engines, and more. These are thing that we do interact with and use frequently. You'd be hard pressed to find someone who has never had anything in their life affected by jet engines and modern aviation, be it through travel or shipping things. Same with a microwave, many people have interacted with them. A competition, not necessarily between two companies, but rather two halves of the world, did indeed produce new commodities which would help many people in the future.

The song, "Song about Life in Virgina" is about a female indentured servant who shows as a glimpse into 5 years of her life while in that time. This woman writes very minimal about what she went through while telling a lot. The woman tells us “Five Years served I, Under master guy, In the land of Virginny, O: which made me for to know, sorrow, grief, and woe”, this one part shows us without explanation that she gave 5 years of her life taking care of this master needs to make his life better to have to live a like of misery for herself. Indentured servants are those who work for a person in return for things like food and shelter and no pay, this woman in this poem shows that through out her time she had to work and live in bad conditions like little food, thin clothing, dirty areas to sleep in, etc. This shows us as readers that these people that were indentured servants gave many years of their lifes to work for these masters which make their lifes easier and better just to have to live in horrible conditions to get food, clothing, and shelter.

I love how these questions get straight to the point, but they will be beneficial for us as educators. I think when teaching in the arts, things will always have to change and be explained. These questions address them, but also are not accusatory which is also not beneficial to objectively understanding.

I appreciate having these steps to help students through the creative process. It is so important that students take ownership of their creativity, but I have often asked, " How do you do this? These steps really help lay it out and help us know how to motivate them.

Constrants represent context, and generative AI has problems observing and identifying context.

Holy real trump parallel and they dont even know it

Greivence

because the unions are gone

But labour unions are no longer mobilizing the other populations

Less democratic voting by extension

delaying the effect of deindustrialization

Which all eroded as the midwest become non-defined by industrialism

And the unification of races around labor would have eroded

and fair enough, but shouldn,t they attach that to both parties, or to specific policies

And in that wake, the institutions that once supported the industrial sector were no longer relevant

Midwest city was literally being robbed of its livelhood

Industry

Which was not happening in the city

The midwest is (understandably) butt hurt

His policies hurt the people of the midwest besides just ignoring them

i.e. race

Dark

Midwest to coast shift

And its contunied fleeing and inequality compared to the coast

And also as professionals defect to the democratic larty, one with clear political implications

Getting worse and nobody cares

Because trump remains at least in part decided by race while the hostility towards democrats is more ubiquitsly economic

White people are affected too

Because whites had something to fall back on

Outlandish: Well yes? isn't that why there are so many genres to choose from? if a person thinks that it's not enough then pick something else. Why does there have to be hate and finding something threatening?

Outlandish: I wholeheartedly disagree with this take on games. I believe that a game doesn't have to be complex or even hard to be considered interesting. Different people have different views on what's considered interesting: there are people who'd prefer easier and light hearted games.

Outlandish: "walking simulator" being used as an insult is so stupid, like why would you be offended by that at all

In my opinion, games typically considered walking simulators are best when they do what's described in this section. I honestly think this is the only kind of story which walking simulators excel at (stories in which the horrible truth about the main character is revealed slowly).

Outlandish: This interpretation of this misleading horror element of the game 'Gone Home' is interesting for sure. But, I do wonder if the creators of the game meant for it to be intentional because I believe a majority of players felt this way.

Outlandish: It's more than easy to lose sight of what you're doing and not realize what the game is going for, I remember when I tried to play video games as a kid I wouldn't realize what I was actually supposed to do and then get mad when nothing was going my way. I felt that way when playing Gone Home at times.

Outlandish: Not sure if this qualifies as outlandish, but if this is referring to the old Atari video game, I dont think it was text.

Outlandish: If there is no hierarchy of skill, then there is no clear status structure, which might threaten players who value mastery. It makes me rethink complaints about “no gameplay” as possibly complaints about “no competition.”

OUTLANDISH .I think this is pretty stupid. I can get walking out in nature or like in real life but on a screen I'm not gonna be admiring a video game the same way I admire nature even if it's super realistic. Like it's still a game and though there could be aspects of beauty in no way do I believe I am gonna retain my humanity.

Outlandish I found this claim crazy, adding depth I had never considered to games with "no consequences". In this way, it contradicts itself. There are consequences. They' re knowing what happened is fully your fault. If anything, the consequence is more scarring than any "death" could be.

Outlandish - Personally, I think I would react the same if a game I spent hundreds of hours making got grouped into a category of games that's considered to be trash by the general public.

Outlandish: I'm surprised that someone defending walking simulators would admit that they're boring to their critics. It seems impossible to deny that they aren't as excited as action-based games, so most of the debate centers around the strength of their message instead.

OUTLANDISH: Permalife is not difficult. If the player is always alive, there are no stakes and nothing to lose. The player's instincts to run if they are seen or fight back or hide are completely neutralized. This does not benefit the player in any way. There is nothing to deal with in the face of survival because survival is guaranteed.

Outlandish: Then what's the point? Walking sims bore me without having an objective. Feels mindless.

I think it's interesting to see how participating in a walking simulator game allows you to view a story from a completely different perspective that you might be used to. In terms of Gone Home, the player is detached in terms of knowing what has been happening in their home and family across the time period they have been gone, that presents the opportunity for judgement and other emotions to arise.

This quote really makes me think about the tension between immersion and observation in games. Gone Home lets the player witness an important queer story without ever participating in it…does that create empathy, or does it keep the player at arm’s length? It also raises questions about what it means to “experience” a story through a character: can understanding and reflection replace direct involvement, or does passivity limit the emotional impact?

The way that the player become an intruder in your own home is really crazy to think about. Personally, for me it can't be a home if you don't want to be there and you don't even recognize all the character traits it makes as a home. This "long absence" that's there is something that can make it feel like are an intruder but it should be quick turnaround to feel like you're home. This makes you realize that something is really wrong in this home and that's really the horror aspect of this game.

I do get what they’re saying, but I’m not fully convinced that walking is automatically grounds for reflection. Although slowing down can create space to think, it just ends up becoming boring for the player if there is nothing big happening. If the slowness of a game is intentional, like it is in walking simulators, then the world has to carry a lot of weight and substance.

This idea is important because it refutes the notion that games typically considered walking simulators are simply equivalent to action games without the enemies. Walking simulators place the emphasis of their interactive aspect on the features of the world around the player.

While I was playing Gone Home, I found it hard to see what the protagonist's role in the story was. Ultimately, I think their main purpose was to serve as a judge for the family's situation, since they are the most separated from Sam's issues with her parents. Similarly, our job as a player is to assess whether or not Sam made a good decision, which is why this ending is so impactful to the player.

This argument can definitely be backed by my own prior experiences. Whether it’s Gone Home or other similar games that I've played and seen–some with horror and mystery aspects–I never truly explored the environments simply for the sake of exploring my surroundings. Rather, I was always driven by a sense of curiosity to unfold the mysteries surrounding my character and the others around me.

Personally dislike this. If I want to retain humanity and walk into unseen places, why do it on a screen? Would much rather go outside and take a walk. A proper mental reset turning off screens.

When I read this portion of this passage, I was wondering what was the point of being so hateful towards alternative types of media? I feel that It is odd for gamers to be so affected by different types or games entering the gaming sphere. Shouldn't they be happy that something they love is gaining more traction and new ideas are being implemented?

I think this point of adventure games making it easy to move to the interesting parts is something I see fairly often. There's not many games I’ve played that don't have some sort of feature to skip to the interesting parts and skip over travel. I have noticed that most games do require you to usually travel to a particular location the more time-consuming way at least once before you can skip there immediately.This definitely allows the player to appreciate the action of exploration more.

The ability to interact with objects further elevates the story and environmental atmosphere of 'Gone Home', which made the walking aspect of the game much more meaningful to me.

I think this idea of Permalife is the most interesting idea in this chapter. Most games are focused on survival mechanics where death is the reset button. But permalife suggests that the challenge isn’t dying by continuing. It mirrors life, and that allows for emotional issues to really stand out through these games. But I also think that there needs to be something to keep the player moving forward. Sure some people may go on without a failure condition, but others would feel unmotivated to do so. They would need to nail down aspects like emotional weight, narrative curiosity and more for the game to really work.

I find it interesting that calmer games like walking sims originated from more violent and action-packed games like first-person shooters. Wanting to explore a world usually unavailable due to actions and conflict is common in human beings. It’s like setting a pack of cookies on a table, saying no one can eat it, and then leaving. Surely no one will miss one cookie in the pack. It’s the same concept for these shooter games. Players are busy running around, surviving, and fighting, so they don’t get to actually appreciate the world they are in (by design). It makes a part of their brain curious to experience the world they are already in without the pressures of battle, so a modder will take a cookie form the pack and eat it, opening up the rest of the pack for other people to enjoy. That’s when they realize the game they were praising for graphics and immersion is actually just a rough sketch, raisin cookies when they thought they were chocolate chips. This creates a sense of disappointment. I can see how walking sims were born from it. To cure the disappointment players felt when they realize the game they loved isn’t as polished as they thought. But without the allure of fighting, walking sims need something else, some other temptation. So, they promise ice cream with the cookies, sprinkling lore and stories into their world. Finding out pieces of the story give players their dopamine boost while satisfying their curiosity for adventure. The article then mentioned punishments within the game and how walking sims remove that and instead explore living with the consequences of your actions. This adds more depth to the ice cream and cookies players have been enjoying before, forcing them to either love or hate them more intensely.

Originally, I was not a fan of the subversion of my expectations, but in the past week since I have played it and discussed the game in class, the horror elements have grown on me. Over time, I have started to realize the purpose of subverting my expectations. The realization that Katie is too late to help her sister is cleverly implemented. Seeing satanic imagery leads the player to believe in supernatural elements, but it is just a red herring. The fear the player has allows them to relate to Sam and her experience of coming out. The lightning and creaking noises make the player anxious but not so anxious that they can’t keep playing and have to take a break. As this feeling grows, it transforms into internal fears and intensifies the feeling for the player.

People would never have thought of first-person shooters such as Doom and Unreal with walking simulators like Gone Home, but if you take out the violence in FPS games they are walking sims. Especially in open world games like Zelda you walk around the majority of the time. The only difference is that there's fights from time to time while walking sims tell stories through the environment.

Walking simulators challenge the traditional view that games were designed only for players to win. The game’s purpose shifts from “Achieve a certain number of kills” or “Capture a certain number of enemy bases” to “Explore the house” or “Check your surroundings.” Players learn how to interpret and examine their environments.

It is interesting to me that there were mods created for Quake that removed the entire main goal of the game. I have never thought about taking out a main mechanic of a videogame and then playing it. It makes me rethink all the games ive played and imagine it just like exploration. Playing games with different music can change the whole vibe of the game, I have noticed, like i remember playing horror games with my own music and it made it not as scary.

Goes against the idea that writers write well due to their spark within them. The author goes against this belief that many people have due to romanticization. Writers work hard to get their work done; it is unrealistic to believe that this is not the case.

I am someone who has been exposed to procrastination, especially when it comes to my schoolwork. I push all my work until the last minute, causing me to rush to get it done in the end.

Drinking can relieve writers of any pressure or anxiety they are feeling by doing this so that the writing can come out more natural.

It is not always about being deprived of sleep; writer's block and procrastination can come from psychological issues such as anxiety or depression. It can also come from social and physical factors.

Nathan Mortensen

hypothe.sis intro

Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

Learn more at Review Commons

Reply to the reviewers

General Response to Review

We would like to thank all three reviewers for their encouraging comments on our manuscript. We now submit our revised study after considerable efforts to address each of the reviewer concerns. I will first provide a response related to a major change we have made in the revision that addressed a concern common to all three reviewers, followed by a point-by-point response to individual comments.

Replacing LRRK2ARM data with a LRRK2 specific type II kinase inhibitor: The most critical issue for all 3 reviewers was the use of our new CRISPR-generated truncation mutant of LRRK2 that we called LRRK2ARM. We had not provided direct evidence of the protein product of this truncation, which was a significant limitation. To address this we performed proteomics analysis of all clones, and to our surprise, we identified 7 peptides that were C-terminal to our "predicted" stop codon we had engineered into the CRISPR design. A repeat of the deep sequencing analysis in both directions then more clearly revealed site specific mutations leading to 4 amino acid changes at the junction of exon 19, without introducing a stop codon. Given that we could not detect the protein by western blot (even though proteomics now indicated the region of LRRK2 recognized by our antibodies was present) we decided to remove this clone from the manuscript. In the meantime we had compared the ineffectiveness of MLi-2 to block Rab8 phosphorylation during iron overload in the LRRK2G2019S cells with a type II kinase inhibitor called rebastinib. The data showed very clearly that treatment with rebastinib reversed the iron-induced phospho-Rab8 at the plasma membrane (and by western blot, in new Fig 3). Since this inhibitor is very broad spectrum inhibiting ~30% of the kinome we reached out to Sam Reck-Peterson and Andres Leschziner, experts in LRRK2 structure/function, who recently developed a much more selective LRRK2-specific type II kinase inhibitor they called RN341 and RN277 (developed with Stefan Knapp PMID: 40465731). These compounds effectively coupled the MLi-2 compound through an indole ring to a rebastinib type II compound to provide LRRK2 binding specificity to the efficient DYG "out" type II inhibitor. As with rebastinib, the new LRRK-specific kinase inhibitors also effectively reversed the cell surface p-Rab8 seen in LRRK2G2019S, iron loaded cells. These new data provide the first biological paradigm where the kinase activity of LRRK2 is resistant to type I MLi-2, yet remains highly sensitive to type II inhibitors. While the loss of our LRRK2ARM clone marks a significant change in the manuscript we believe the main message is stronger with the addition of the new LRRK2 specific type II kinase inhibitor. Our data show that it is indeed the active kinase function of LRRK2G2019S that is impacting the iron phenotypes we observe but highlight the conformational specificity upon iron overload such that MLi-2 is ineffective. The overall phenotypes we observe in LRRK2G2019S macrophages remain unchanged and are now expanded within the manuscript. We hope reviewers will agree that our work provides important new insights into LRRK2 function in iron homeostasis while opening new avenues of research in future studies.

Given this new information we have changed the title from "LRRK2G2019S acts as a dominant interfering mutant in the context of iron overload" to the more accurate "LRRK2G2019S interferes with NCOA4 trafficking in response to iron overload leading to oxidative stress and ferroptotic cell death."

Response to Reviewer 1

Reviewer 1 (R1): There are two major concerns with the data in their present form. In brief, first, the G2019S cells express much less LRRK2 and more Rab8 that the WT cells and this severely affects interpretability.

Heidi McBride (HM): We agree that the LRRK2G2019S lines express lower levels of LRRK2 than wild type, which is a previously documented phenomenon, presumably as the cell attempts to downregulate the increased kinase activity by reducing protein expression. However, the levels of Rab8 across 10s of experiments do not consistently show any differences between the wild type, G2019S and KO. We have provided more comprehensive quantifications of the blots in the revised version, and the Rab8 levels are consistent across all the blots presented in the manuscript (Figure 1A and 1B).

R1: Second, the investigators used CRISPR to truncate the endogenous LRRK2 locus to produce a hypothetical truncated LRRK2-ARM polypeptide. This appears to have robust effects on NCOA4, in particular, which drives the overall interpretation of the data. However, the expression of this novel LRRK2 species is not confirmed nor compared to WT or G2019S in these cells (although admittedly the investigators did seek to address this with subsequent KO in the ARM cells). It would be premature to account for the changes reported without evidence of protein expression. This latter issue may be more easily addressed and could provide very strong support for a novel function/finding, see more detailed comments below, most seeking clarifications beyond the above.

HM: As described in my common response above, we have removed the LRRK2ARM data from the manuscript.

R1: Need to make clear in the results whether the G2019S CRISPR mutant is heterozygous or homozygous (presumably homozygous, same for ARM)

HM: The RAW cell line we generated is homozygous for the G2019S and the KO alleles. We added this to the beginning of the results section and methods.

R1: The text of the results implies that MLi2 was used in both WT and G2019S Raw cells, but it's only shown for G2019S. Given the premise for the use of RAW cells, it's important to show that there is basal LRRK2 kinase activity in WT cells to go along with its high protein expression. This is particularly important as the G2019S blot suggests minor LRRK2-independent phosphorylation of Rab8a (and other detected pRabs). One would imagine that pRab8 levels in both WT and G2019S would reduce to the same base line or ratio of total Rab in the presence of MLi2, but WT untreated is similar to G2019S with MLi2. This suggests no basal LRRK2 activity in the Raw cells, but I don't think that is the case.

HM: We have included the data from MLi-2 treatment of wild type cells in Fig 3C quantified in D. Again, the baseline levels of Rab8 are unchanged across the genotypes. However, the reviewer is correct that there is some baseline LRRK2 kinase activity that is sensitive to MLi2 in wild type cells. This is seen most clearly on the autophosphorylation of LRRK2 at S1292 in Fig 3C. The pRab8 blots is not as clear in wild type cells. It is likely that LRRK2 must be actively recruited to membranes (as seen by others with LLOME, etc) to easily visualize p-Rabs in wild type cells. Nevertheless, we do clearly see the activity of autophosphorylation in wild type cells. Therefore while we understand the reviewers point that there should be some Rab8 phosphorylation in wild type cells, we don't see a significant, or very convincing, amount of it in our RAW macrophages.

R1: Also, in terms of these cells, the levels of LRRK2 are surprisingly unmatched (Fig 1A, 1D, 1H, S1D, etc.) as are total levels of Rab8 (but in opposite directions) between the WT and G2019S. This is not mentioned in the Results text and is clearly reproducible and significant. Why do the investigators think this is? If Rab8 plays a role in iron, how do these differences affect the interpretation of the G2019S cells (especially given that MLi2 does not rescue)? Are other LRRK2-related Rabs affected at the protein (not phosphorylation level)? Could reduced levels of LRRK2 or increase Rab 8 alone or together account for some of these differences? Substantial further characterization is required as this seriously affects the interpretability of the data. Since pRab8 is not normalized to total Rab8, this G2019S model may not reflect a total increase in LRRK2 kinase activity, and could in fact have both less LRRK2 protein and less cellular kinase activity than WT (in this case).

HM: In our hands, the RAW cells with homozygous LRRK2G2019S mutations show clearly that the total protein levels of LRRK2 is reduced compared to wild type, which is likely a compensatory effect to reduce cellular kinase activity overall. We understand that some of our previous blots were not so clear on the total Rab8 levels across the different experiments. We have repeated many of these experiments and hope the reviewer can see in Figs 1A, 3C, 3E, 3J, and Sup3A that the total Rab8 levels are stable across the conditions. We also present quantifications from 3 independent experiments normalizing the pRab8/Rab8 levels in all three genotypes in untreated and iron-loaded conditions (Supp Fig 3A and B), and upon MLi2 treatment (Fig 3C). In 3C and D the data show the effectiveness of MLi-2 to reduce pRab8 in control conditions, but the resistance to MLi-2 in FAS treated cells.

R1: Presumably, the blots in 1H are whole cell lysates and account for the pooled soluble and insoluble NCOA4 (increased in G2019S), as there is no difference in soluble NCOA4 (Fig 2H). I suspect the prior difference is nicely reflected in the insoluble fraction (Fig 2H). This should be better explained in the Results text. This is a very interesting finding and I wonder what the investigators believe is driving this phenotype? Is the NCOA4 partitioning into a detergent-inaccessible compartment? Does this replicate with other detergents, those perhaps better at solubilizing lipid rafts? Is this a phenotype reversible with MLi2? Very interesting data.

HM: We apologize for not being clearer in the text describing the behavior of NCOA4. The reviewer is correct that the major change in G2019S is the increased triton-X100 insoluble NCOA4. Previous work has established that NCOA4 segregates into detergent-insoluble foci upon iron overload as a way to release it from ferritin cages, and this fraction is then internalized into lysosomes through a microautophagy pathway (see Mizushima's work PMID: 36066504). In Fig 1I we show that the elevation in NCOA4 and ferritin heavy chain seen in untreated G2019S cells can be cleared upon iron chelation with DFO, indicating that the canonical NCOA4 mediated ferritinophagy (macroautophagy) pathway remains intact to recycle the iron in conditions of iron starvation. However in Figure 2 we show that conditions of iron overload, when NCOA4 segregates from ferritin (to allow cytosolic storage of iron), this form of NCOA4 cannot be degraded within the lysosome through the microautophagy pathway, and begins to accumulate. We see this with our live and fixed imaging compared to wild type cells (Fig 2A,D), and by the lack of clearance seen by western blot (Fig 2E). As for the impact of MLi-2, we observe some reversal of NCOA4 accumulation in untreated cells at 4 and 8 hrs after MLi-2 treatment (Supp Fig 2F). However, in iron loaded conditions the high NCOA4 levels in G2019S cells are MLi2 insensitive, while the elevated NCOA4 in wild type cells is reduced upon MLi2 addition (Fig. 2F, compare lates 3vs4 in wt with lanes 7vs8 in G2019S). This is consistent with a block in the microautophagy pathway of phase-separated NCOA4 degradation in G2019S cells.

R1: Figure 2 describes the increased NCOA4-positive iron structures after iron load, but does not emphasize that the G2019S cells begin preloaded with more NCOA4. How do the investigators account for differential NCOA4 in this interpretation? Is this simply a reflection of more NCOA4 available in G2019S cells? This seems reasonable.

HM: The reviewer is correct, we showed that there is some turnover of NCOA4 in untreated conditions through canonical ferritinophagy, but in iron overload this appears to be blocked, the NCOA4 segregates from ferritin and remains within insoluble, phase-separated structures that cannot be degraded through microautophagy. We have written the text to be more clear on these points.

R1: These are very long exposures to iron, some as high as 48 hr which will then take into account novel transcriptomic and protein changes. Did the investigators evaluate cell death? Iron uptake would be trackable much quicker.

HM: We agree that many things will change after our FAS treatments and now provide a full proteomics dataset on wild type and G2019S cells with and without iron overload, which is presented in Figure 4A-B. Indeed Figure 4 is entirely new to this revised submission. The proteomics highlighted a series of cellular changes that reflect major cell stress responses including the upregulation of HMOX1 (western blots to validate in Supp Fig 4A), an NRF2 transcriptional target consistent with our observation that NRF2 is stabilized and translocated to the nucleus in G2019S iron loaded cells (Sup Fig 4B,C). There are several interesting changes, and we highlighted the three major nodes, which are changes in iron response proteins, lysosomal proteins - particularly a loss of catalytic enzymes like lysozymes and granzymes consistent with the loss of hydrolytic capacity we show in Fig. 4C,D. We also noted changes in cytoskeletal proteins we suspect is consistent with the "blebbing" of the plasma membrane we see decorated with pRab8 in Fig 3. To test the activation of lipid oxidation likely resulting from the elevation in Fe2+ and oxidation signatures we employed the C11-bodipy probe and observe strong signal specific to the G2019 iron-loaded cells, particularly labelling endocytic compartments and the cell surface (Fig. 4E-G).

Lastly, an analysis of SYTOX green uptake experiments was done to monitor the uptake of the dye into cells that have died of cell membrane rupture, commonly used to examine ferroptotic cell death. We now show the G2019S cells are very susceptible to this form of death (Fig 4H,I). These data add new functional evidence for the consequence of the G2019S mutation in an increased susceptibility to iron stress.

R1: The legend for 2F is awkward (BSADQRED)

HM: We have changed this to BSA-DQRed, which is a widely used probe to monitor the hydrolytic capacity of the lysosome.

R1: Why are WT cells not included in Fig 2G?

HM: We have now included new panels in Fig 3C,D showing wild type and G2019S +/- FAS and +/-ML-i2 with quantifications of pRab8/Rab8.

R1: The biochemical characterization of NCOA4 in the LRRK2-arm cells is a great experiment and strength of the paper. The field would benefit by a bit further interrogation, other detergents, etc.

HM: We have removed all of the LRRK2ARM data given our confusion over the impact of the 4 amino acid changes in exon 19 and our inability to monitor this protein by western blot. The concept that NCOA4 enters into TX100 insoluble, phase separated compartments has been well established, so we didn't explore other detergents at this point.

R1: Have the investigators looked for aberrant Rab trafficking to lysosomes in the LRRK2-arm cells? Is pRab8 mislocalized compared to WT? Other pRabs?

HM: We did initially show that pRab8 was also at the plasma membrane in the LRRK2ARM cells, and we still focus on this finding for the G2019S, seen in Fig 3A,B,F,H. We did try to look at other p-Rabs known to be targets of LRRK2 but none of them worked in immunofluorescence so we couldn't easily monitor specific traffic and/or localization changes for them.

R1: The expression levels and therefore stability of the ARM fragment is not shown. This is necessary for interpretation. While very intriguing, the data in Aim 3 rely on the assumption that the ARM fragment is expressed, and at comparable levels to G2019S to account for phenotypes. The generation of second clone is admirable, but the expression of the protein must be characterized. This is especially true because of the different LRRK2 levels between WT and G2019S. One could easily conceive of exogenous expression of a tagged-ARM fragment into LRRK2 KO cells, for example, as another proof-of-concept experiment. If it is truly dominant, does this effect require or benefit from some FL LRRK2? It seems easy enough to express the LRRK2-ARM in at least WT and KO RAW cells.

HM: We agree and our attempts to understand this clone resulted in its removal from the manuscript. We did also express cDNA encoding our ARM domain (up to exon 19), but it didn't phenocopy the CRISPR clone, which of course made sense once we had better proteomics and repeated our deep sequencing.

In our further efforts to understand why our phenotype was MLi-2 resistant upon iron overload we expanded to examine the impact of pan-specific TypeII kinase inhibitors, and then reached out to the Reck-Peterson and Leschziner labs to obtain a newly developed LRRK2 selective type II kinase inhibitor. These all very efficiently reversed the pRab8 signals seen at the plasma membrane of G2019S cells upon iron overload (Fig 3E-K). Therefore the G2019S is not dominant negative, as we had initially supposed, rather there is a specific conformation of LRRK2 in high iron that potentially opens the ATP binding pocket to bind the type II inhibitors, but not MLi2. We do not understand exactly what this conformation is but likely involves new protein interactions specific to high iron, or perhaps LRRK2 binds iron directly as a sensor somehow that ultimately leads to the differential sensitivity we observe between type I and type II kinase inhibitors. Our data indicate that MLi-2 treatment in clinic will not be protective against iron toxicity phenotypes that may contribute to PD, where these newer selective type II LRRK2 kinase inhibitors would be effective in this conformation-specific context of iron toxicity.

R1: Does iron overload induce Rab8a phosphorylation in a LRRK2 KO cell? This would be a solid extension on the ARM data and support the important finding that an additional kinase(s) can phosphorylate Rab8a under these conditions, and while not unexpected, this may not have been demonstrated by others as clearly. It also addresses whether the ARM domain is important to this other putative kinase(s), which may add value to the authors' model.

HM: Iron overload does not induce pRab8 in LRRK2 KO cells, as seen by immunofluorescence in Fig 3A,B, and western blot in Supp Fig 3 A,B. With our new type II kinase inhibitor data we can confirm that the plasma membrane localized Rab8 is indeed phosphorylated by LRRK2.

R1: Minor concern - the abstract but not the introduction emphasizes a hypothesis that loss of neuromelanin may promote cell loss in PD (through loss of iron chelation), while post mortem studies are by definition only correlative, early works suggested that the higher melanized DA neurons were preferentially lost when compared to poorly melanized neurons in PD. This speculation in the abstract is not necessary to the novel findings of the paper.

HM: We appreciate that the links to iron in PD are correlative, we have maintained some of our discussion on this point within the manuscript given the lack of attention the field has paid to the cell biology of iron homeostasis in PD models. If there is a cell autonomous nature to the loss of DA neurons in PD, iron is very likely to be a part of this specificity in our opinion. Most of the newer MRI studies looking at iron levels in patient brains are showing higher free iron and working on this as potential biomarkers of disease. The precise timing of this relative to the stability/loss of neuromelanin is, I agree, not really clear.

R1: (Significance (Required)): This study could shed light on a both novel and unexpected behavior of the LRRK2 protein, and open new insights into how pathogenic mutations may affect the cell. While studied in one cell line known for unusually high LRRK2 expression levels, data in this cell type have been broadly applicable elsewhere. Give the link to Parkinson's disease, Rab-dependent trafficking, and iron homeostasis, the findings could have import and relevance to a rather broad audience.

HM: We are so very appreciative that reviewer 1 feels our work will be of interest to the PD and cell biology communities.

Response to Reviewer 2

Reviewer 2 (R2): Major: Please confirm that the observed phenotype is conserved within bone marrow-derived macrophages of LRRK2 G2019S mice. These mice are widely available within the community and frozen bone marrow could be sent to the labs. The main reason for this experiment is that CRISPR macrophage cell lines do sometimes acquire weird phenotypes (at least in our lab they sometimes do!) and it would strengthen the validity of the observations.

HM: We did a series of experiments on primary BMDM derived from 3 pairs of wild type, LRRK2G2019S and LRRK2KO mice. We examined levels of ferritin heavy and light chains in steady state and withFAS treatment experiments. Unfortunately the data did not phenocopy the RAW macrophage lines we present here since FTL and FTH were mostly unchanged. We did observe an increase in NCOA4 levels, consistent with potential issues with microautophagy as observed in our RAW system.

While we understand the danger that our phenotypes are nonspecific and linked to a CRISPR-based anomaly, there are a number of arguments we would make that these data and pathways are potentially very important to our understanding of LRRK2 mutant phenotypes and pathology. The first point is that we now include a LRRK2-specific type II kinase inhibitor that reverses the iron-overload pRab8 accumulation at the plasma membrane in LRRK2G2019S cells, showing that this is at least directly linked to LRRK2 kinase activity, even though it is resistant to MLi2.

Second, Suzanne Pfeffer recently published their single cell RNAseq datasets from brains of untreated LRRK2G2019S mice (PMID: 39088390). She reported major changes in Ferritin heavy chain (it is lost) in very specific cell types of the brain, astrocytes, microglia and oligodendrocytes, with no changes in other cell types at all (her Fig 6 included left). This is consistent with a very context specific impact of LRRK2 on iron homeostasis that we don't yet understand.

Third, the labs of both Cookson, Mamais and Lavoie have been working on the impact of LRRK2 mutations on iron handling in a few different model systems, including iPSCs, and see changes in transferrin recycling and iron accumulation. Those studies did not go into much detail on ferritin, NCOA4 and other readouts of iron homeostasis but are roughly in agreement with our work here. In the last biorxiv study submitted after we sent this work for review they concluded their phenotypes were reversed by MLi2 treatment, however they required 7 days of treatment for a ~20% restoration in iron levels. Given our work it would seem the impact of LRRK2G019S in high iron conditions is also very resistant to MLi2 treatment. In all these studies we do not yet know for sure whether iron overload in the brain may be a precursor to DA neuron cell death, which could be exacerbated in G2019S carriers. But we hope the reviewer will agree that our approach and findings will be useful for the field to expand on these concepts within different models of PD.

R2: Minor comments: Supplementary Fig 1: I don't think one should normalize all controls to 1 and then do a statistical test as obviously the standard deviation of control is 0.

HM: We agree with the reviewer that statistical testing is not appropriate when the WT control is fixed to a value of 1, as this necessarily eliminates variance in that group; accordingly, we have removed both statistical comparisons and standard deviation from the WT control while retaining variability measures for all experimental conditions. Raw densitometry values could not be pooled across independent experiments due to substantial inter-blot variability, and therefore normalization to the WT control was used solely to allow relative comparison within experiments, acknowledging the inherent quantitative limitations of Western blot densitometry. Ultimately the magnitude of the changes relative to the control lanes in each biological replicate was consistent across experiments, even if the absolute density of the bands between experiments was not always the same.

R2: The raw data needs to be submitted to PRIDE or similar.

HM: All of our data is being uploaded to the GEO databases, protocols to protocols.io and raw data deposited on Zenodo site in compliance with our ASAP funding requirements and the journals.

R2: Some of the western blots could be improved. If these are the best shown, I am a little concerned about the reproducibility. How often has they been done?

HM: We now ensure there is quantification of all the blots for at least 3 independent experiments and have worked to improve the quality of them throughout the revision period.

R2: (Significance (Required)): Considering the importance of LRRK2 biology in Parkinson's and the new biology shown, this paper will be of great interest to the community and wider research fields.

HM: We are so very grateful that the reviewer appreciates that the LRRK2 and PD community will find our work of interest. We hope our revisions will prove satisfactory even in the absence of ferritin changes in primary G2019S BMDM.

Response to Reviewer 3

Reviewer 3 (R3): What is missing in the study is the physiological relevance of these findings, mainly whether this effect actually results in higher cell death during iron overload. Since iron overload is known to result in ferroptosis, it is surprising that the authors have not checked whether the LRRK2 G2019S and ARM cells undergo more ferroptosis relative to LRRK2 WT cells.

HM: We thank the reviewer for pushing us to monitor the functional implications of the iron mishandling upon iron overload in the G2019S RAW cell system. We now add a completely new Figure 4 to get to these functional points. We employed two tools to look at established aspects of ferroptosis, first the C11-bodipy probe that labels oxidized lipids and we see significant signals specific to the G2019S iron loaded cells, where it labels endocytic membranes and the cell surface (Fig 4 E-G). This is consistent with the elevation of free iron 2+. We also used the SYTOX green death assay where the dye is internalized into cells when the cell surface is ruptured and show that G2019S cells die upon iron overload, but not the LRRK2KO or wild type cells (Fig 4 H,I). Lastly, we performed full proteomics analysis of the wt and G2019S RAW cells in iron overload conditions. These data provide a better view of the full stress response initiated in the G2019S cells, including the upregulation of HMOX1 (an NRF2 target gene), changes in lysosomal hydrolytic enzymes consistent with the reduction in BSA-DQRed signals, and in cytoskeleton, which is consistent with the plasma membrane blebbing phenotypes we see in G2019S (Fig. 4A-D and Supp. Fig 4 data). We hope these new data help to position the phenotype into a more physiological output.

R3: Moreover, their conclusion of the findings as "resistant to LRRK2 kinase inhibitors" is not convincing, since in most of the studies, they have removed the kinase domain, and this description implies the use of pharmacological kinase inhibition which has not been done in this paper.

HM: We took this comment to heart and, as explained in the general response we removed the LRRK2ARM clones from the study. To understand the kinase function in the iron overload conditions we first explored the pan-specific type II kinase inhibitor rebastinib, shown to inhibit LRRK2. In contrast to MLi2, this drug effectively blocked p-Rab8 in G2019S cells exposed to high iron. However, since it is not specific and likely inhibits about 30-40% of all kinases we reached out to the Reck-Peterson and Leschziner labs who have developed a LRRK2 specific type II kinase inhibitor (published in June 2025 PMID: 40465731). They provided these to us (along with a great deal of discussion) and the two drugs both blocked the effect of LRRK2G2019 on p-Rab8 at the plasma membrane. These data show that the phenotypes we observe are indeed linked to the increased kinase activity of LRRK2, even though they are fully resistant to MLi-2. It suggests that high iron results in some alteration in LRRK2 conformation that alters the ability of MLi2 to block the kinase activity, while still allowing the type II kinase inhibitors that bind deeper in the ATP-binding pocket, to functionally block activity. We believe that these new data remove a great deal of confusion we had in the initial submission to explain the MLi-2 resistance.

R3: There is lower LRRK2 expression in LRRK2 G2019S cells, have the authors checked Rab phosphorylation to validate the mutation?

HM: We agree that the G2019S mutation leads a reduction in total LRRK2 levels in the cell, which is likely a compensatory effect to lower kinase activity in the cell. We do show that the G2019S mutation has clear activation of phosphorylation on both Rab8 and at the autophosphorylation site S1292 of LRRK2, as seen in Fig 1A, quantified in Fig 1B. In untreated conditions, these phosphorylation events are reversible upon treatment with MLi-2. We also provide the sequencing data in the supplement to confirm the presence of the G2019S mutation in this clone, shown in Supp Fig. 1A.

R3: The authors should specify if their cells are heterozygous or homozygous since they are discussing a dominant interfering mutant.

HM: The G2019S and LRRK2 KO are both homozygous. We state this early in the results section and the methods.

R3: The transferrin phenotype validated through proteomics and western blot is solid. HM: We agree, thank you very much!

R3: Quantification in figure 1F-G is problematic, not clear what they mean by "diffuse and lysosomal". Puncta is either colocalising with lysosomes or not colocalising. This needs to be clarified and re-analysed.

HM: We apologize for the confusion. In control cells the Cherry tagged FTL is efficiently cycling through the lysosomes and we don't see a strong cytosolic (diffuse) pool, which likely reflects the relatively iron-poor culture conditions. However, in G2019S cells, there is a highly elevated amount of FTL, with a strong cytosolic/diffuse stain in steady state, with some flux into lysosomes. In this experiment we chelated iron to test whether this cytosolic pool of FTL was capable of clearing through the lysosomes (ferritinophagy). While there is a cytosolic (diffuse) pool that remains, the pool that fluxes into the lysosome increases in G2019S chelated cells. This is also seen by the reduction in total FTL seen by western blot (endogenous FTL). Our conclusion here is that the general ferritinophagy machinery remains functional in G2019S cells. We have changed the term "diffuse" to "cytosolic" and improved our description of this experiment in the text.

R3: Text in the first results part called "LRRK2G2019S RAW macrophages have altered iron homeostasis" is very long. It could be divided into more sections to improve readability. HM: We have improved the text to be more descriptive of the conclusions and added new sections

R3: If the effect is armadillo-dependent, where does LRRK2 G2019S is implicated since there is no kinase domain in these cells?

HM: Our new data employing the LRRK2-specific type II kinase inhibitors now confirm that the effects of the G2019S on iron overload are indeed kinase dependent, it's just insensitive to MLi2.

R3: The authors do not show any controls (PCR, sequencing) confirming knockout or truncation. HM: We did higher resolution proteomics and deep sequencing and learned that the "Arm" mutation was not a truncation but a series of 4 point mutations around exon 19. Therefore we removed all data referring to this clone and replaced it with the use of the type II kinase inhibitor experiments. We feel this removed a lot of confusion and provides much clearer conclusions on the role of the kinase activity in iron overload. We may continue to explore what the 4 amino acid mutations created such strong phenotypes, as it could reflect a critical conformational change that impacts the kinase activity. But that is for future work. We now include the sequencing files of the G2019 and KO as Supplementary Data Files 1 and 2.

R3: The data is interesting and the image quality with the insets is very high. HM: We thank the reviewer for their positive comments!

R3: Mutant not clearly described in text, did the authors remove just the kinase and ROC-COR domains or all the domains downstream of the Armadillo domain? This is not clear. HM: We have removed the clone from the manuscript.

R3: The authors cannot conclude that their phenotype is due to the independence of the kinase domain specifically as they are also interfering with the GTPase activity by removing the ROC-COR domains. HM: We agree and our new drugs allow us to confirm that the phenotypes are due to kinase activity, but there is a new conformation of LRRK2 induced in high iron that renders the kinase domain resistant to MLi-2 inhibition. We discuss this in the manuscript now.

R3: In Figure 3E, is the difference between the "ARM CTRL" and the "ARM FAS" conditions significant? A trend appears to be there, but the p-value is not shown. HM: these data are now removed.

R3: In figure 4A, it would have been important to check if Rab8 phosphorylation is also observed in LRRK2 KO cells after administration of FAS to further evaluate the mechanism through which this Rab8 phosphorylation is occurring.

HM: We show that the pRab8 is specific to the G2019S lines and not seen in LRRK2 KO (Fig 3A,B, Supp. Fig. 3A,B).

R3: The vinculin bands in figure 4A are misaligned with the rest of the bands.

HM: We now provide new blots for all of these experiments (in Fig 3) as we removed the LRRK2ARM data from the manuscript and the appropriate loading controls are all included.

R3: The authors do not have any controls to validate the pRab8 staining in IF. This is an important caveat and needs to be addressed. HM: We now include siRNA validation of Rab8 (vs Rab10) to confirm the specificity of the antibody to pRab8 in IF where it labels the plasma membrane in G2019S iron loaded cells.

R3: The authors should have checked if FAS administration in the LRRK2 G2019S and the ARM cells is leading to ferroptotic cell death (or cell death in general). This is key to validate the link between the altered iron homeostasis in LRRK2 G2019S cells and increased cytotoxicity observed during neurodegeneration.

HM: As mentioned above, we have added extensively to our new Fig 4 to include full proteomics analysis of the changes in iron loaded G2019S cells, we use C11-Bodipy probes to monitor lipid oxidation, and SYTOX green assays to monitor cell death through cell surface rupture (consistent with ferroptosis). We thank the reviewer for pushing us to do these experiments and provide further relevance to the potential for LRRK2 mutations to promote cell toxicity during neurodegeneration.

R3: Regarding the literature, the authors are missing some important papers that are preprinted and these studies need to be discussed. This includes a report with opposite findingshttps://www.biorxiv.org/content/10.1101/2025.09.26.678370v1.full and a report showing kinase independent cell death in macrophages https://www.biorxiv.org/content/10.1101/2023.09.27.559807v1.abstract

HM: We thank the reviewers for alerting us to the biorxiv papers, one of which was submitted after we sent our manuscript to review. We are excited to see the growing interest in the impact of LRRK2 function in iron homeostasis and hope our work will contribute to this. Upon reading the study from the LaVoie lab they do show some sensitivity of the iron loaded phenotype in G2019S cells, however they see a ~20% reduction in lysosomal iron after 7 days of MLi treatment in Astrocytes (their Fig 2L). To us, this is very likely an indication of a relatively high resistance to the drug. I'm sure if they tried these new Type II inhibitors the iron load would be much more rapidly reversed. The specificity of their phenotype to Rab8 is also very interesting considering the cell surface localization we see for pRab8 in our iron loaded system. Similar comments for the Guttierez study in macrophages. We have included the findings of these papers within the manuscript and thank the reviewer for pointing them out.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

Referee #3

Evidence, reproducibility and clarity

In this paper, the authors report an interesting phenotype of the LRRK2 G2019S mutation on iron homeostasis in RAW264.7 macrophages. The phenotype is well characterised through proteomic and western blot approaches investigating transferrin and ferritin trafficking. The study is well conducted and data of high quality. The authors also appear to have discovered a cellular context where Rab8 is phosphorylated independently of LRRK2. This is a major finding which can potentially have an important impact in the LRRK2 field. What is missing in the study is the physiological relevance of these findings, mainly whether this effect actually results in higher cell death during iron overload. Since iron overload is known to result in ferroptosis, it is surprising that the authors have not checked whether the LRRK2 G2019S and ARM cells undergo more ferroptosis relative to LRRK2 WT cells. Moreover, their conclusion of the findings as "resistant to LRRK2 kinase inhibitors" is not convincing, since in most of the studies, they have removed the kinase domain, and this description implies the use of pharmacological kinase inhibition which has not been done in this paper.

Significance

Major comments

In Figure 1:

• There is lower LRRK2 expression in LRRK2 G2019S cells, have the authors checked Rab phosphorylation to validate the mutation?
• The authors should specify if their cells are heterozygous or homozygous since they are discussing a dominant interfering mutant.
• The transferrin phenotype validated through proteomics and western blot is solid.
• Quantification in figure 1F-G is problematic, not clear what they mean by "diffuse and lysosomal". Puncta is either colocalising with lysosomes or not colocalising. This needs to be clarified and re-analysed.
• Text in the first results part called "LRRK2G2019S RAW macrophages have altered iron homeostasis" is very long. It could be divided into more sections to improve readability.

In Figure 2:

• If the effect is armadillo-dependent, where does LRRK2 G2019S is implicated since there is no kinase domain in these cells?
• The authors do not show any controls (PCR, sequencing) confirming knockout or truncation.
• The data is interesting and the image quality with the insets is very high.

In Figure 3:

• Mutant not clearly described in text, did the authors remove just the kinase and ROC-COR domains or all the domains downstream of the Armadillo domain? This is not clear.
• The authors cannot conclude that their phenotype is due to the independence of the kinase domain specifically as they are also interfering with the GTPase activity by removing the ROC-COR domains.
• In Figure 3E, is the difference between the "ARM CTRL" and the "ARM FAS" conditions significant? A trend appears to be there, but the p-value is not shown.

In Figure 4:

• In figure 4A, it would have been important to check if Rab8 phosphorylation is also observed in LRRK2 KO cells after administration of FAS to further evaluate the mechanism through which this Rab8 phosphorylation is occurring.
• The vinculin bands in figure 4A are misaligned with the rest of the bands.
• The authors do not have any controls to validate the pRab8 staining in IF. This is an important caveat and needs to be addressed.
• The authors should have checked if FAS administration in the LRRK2 G2019S and the ARM cells is leading to ferroptotic cell death (or cell death in general). This is key to validate the link between the altered iron homeostasis in LRRK2 G2019S cells and increased cytotoxicity observed during neurodegeneration. Regarding the literature, the authors are missing some important papers that are preprinted and these studies need to be discussed. This includes a report with opposite findings https://www.biorxiv.org/content/10.1101/2025.09.26.678370v1.full and a report showing kinase independent cell death in macrophages https://www.biorxiv.org/content/10.1101/2023.09.27.559807v1.abstract

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

Referee #2

Evidence, reproducibility and clarity

In this manuscript the authors describe an interesting connection between the Parkinson's kinase LRRK2 and iron trafficking in RAW macrophages. Expression of the LRRK2 G2029S mutation affects the abundance of ferritin heavy and light chains and therefore the uptake and storage of iron. Interestingly, the loss of the kinase domain still had a strong phenotype, suggesting that this is independent of the kinase function.

The paper is well written and excellently cited. The data is convincing and of good quality.

I have only one request and else very minor comments:

Major: Please confirm that the observed phenotype is conserved within bone marrow-derived macrophages of LRRK2 G2019S mice. These mice are widely available within the community and frozen bone marrow could be sent to the labs.

The main reason for this experiment is that CRISPR macrophage cell lines do sometimes acquire weird phenotypes (at least in our lab they sometimes do!) and it would strengthen the validity of the observations.

Minor comments:

Supplementary Fig 1: I don't think one should normalize all controls to 1 and then do a statistical test as obviously the standard deviation of control is 0. I would normalize to the average of the control, which will provide an error for the control.

The raw data needs to be submitted to PRIDE or similar. This has not happened yet.

Some of the western blots could be improved. If these are the best shown, I am a little concerned about the reproducibility. How often has they been done?

Significance

Considering the importance of LRRK2 biology in Parkinson's and the new biology shown, this paper will be of great interest to the community and wider research fields.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

Referee #1

Evidence, reproducibility and clarity

Goldman et al describe some novel findings with respect to LRRK and iron handling in a series of RAW macrophage cell lines. This cell background is chosen for its recognized high levels of endogenous LRRK2 protein expression, its somewhat broad use in the field, and the investigators add its relevance due to phagocytosis of red blood cells, thus requiring iron robust metabolic processes. Proteomic analyses of WT and G2019S RAW cells revealed multiple iron-related proteins affected by LRRK2 mutation. A deeper candidate-based analysis revealed complex changes in ferritin heavy and light chain and changes in ferric and ferrous iron. Notably, reliable changes in the levels and/or solubility of NCOA4 result from this pathogenic LRRK2 mutation. Unexpectedly, however, these changes were not sensitive to LRRK2 kinase inhibitor treatment. The investigators suggest a dominant effect rather than loss-of-function as subsequent experiments revealed that these effects could be replicated with a LRRK2 variant lacking the kinase domain (LRRK2-ARM) and were not replicated by LRRK2 KO. The data are internally consistent throughout and could certainly shed new important light onto unique and unexpected effects of this LRRK2 mutation.

There are two major concerns with the data in their present form. In brief, first, the G2019S cells express much less LRRK2 and more Rab8 that the WT cells and this severely affects interpretability. Second, the investigators used CRISPR to truncate the endogenous LRRK2 locus to produce a hypothetical truncated LRRK2-ARM polypeptide. This appears to have robust effects on NCOA4, in particular, which drives the overall interpretation of the data. However, the expression of this novel LRRK2 specie is not confirmed nor compared to WT or G2019S in these cells (although admittedly the investigators did seek to address this with subsequent KO in the ARM cells). It would be premature to account for the changes reported without evidence of protein expression. This latter issue may be more easily addressed and could provide very strong support for a novel function/finding, see more detailed comments below, most seeking clarifications beyond the above.

• Need to make clear in the results whether the G2019S CRISPR mutant is heterozygous or homozygous (presumably homozygous, same for ARM)
• The text of the results implies that MLi2 was used in both WT and G2019S Raw cells, but it's only shown for G2019S. Given the premise for the use of RAW cells, it's important to show that there is basal LRRK2 kinase activity in WT cells to go along with its high protein expression. This is particularly important as the G2019S blot suggests minor LRRK2-independent phosphorylation of Rab8a (and other detected pRabs). One would imagine that pRab8 levels in both WT and G2019S would reduce to the same base line or ratio of total Rab in the presence of MLi2, but WT untreated is similar to G2019S with MLi2. This suggests no basal LRRK2 activity in the Raw cells, but I don't think that is the case.
• Also, in terms of these cells, the levels of LRRK2 are surprisingly unmatched (Fig 1A, 1D, 1H, S1D, etc.) as are total levels of Rab8 (but in opposite directions) between the WT and G2019S. This is not mentioned in the Results text and is clearly reproducible and significant. Why do the investigators think this is? If Rab8 plays a role in iron, how do these differences affect the interpretation of the G2019S cells (especially given that MLi2 does not rescue)? Are other LRRK2-related Rabs affected at the protein (not phosphorylation level)? Could reduced levels of LRRK2 or increase Rab 8 alone or together account for some of these differences? Substantial further characterization is required as this seriously affects the interpretability of the data. Since pRab8 is not normalized to total Rab8, this G2019S model may not reflect a total increase in LRRK2 kinase activity, and could in fact have both less LRRK2 protein and less cellular kinase activity than WT (in this case).
• Presumably, the blots in 1H are whole cell lysates and account for the pooled soluble and insoluble NCOA4 (increased in G2019S), as there is no difference in soluble NCOA4 (Fig 2H). I suspect the prior difference is nicely reflected in the insoluble fraction (Fig 2H). This should be better explained in the Results text. This is a very interesting finding and I wonder what the investigators believe is driving this phenotype? Is the NCOA4 partitioning into a detergent-inaccessible compartment? Does this replicate with other detergents, those perhaps better at solubilizing lipid rafts? Is this a phenotype reversible with MLi2? Very interesting data.
• Figure 2 describes the increased NCOA4-positive iron structures after iron load, but does not emphasize that the G2019S cells begin preloaded with more NCOA4. How do the investigators account for differential NCOA4 in this interpretation? Is this simply a reflection of more NCOA4 available in G2019S cells? This seems reasonable.
• These are very long exposures to iron, some as high as 48 hr which will then take into account novel transcriptomic and protein changes. Did the investigators evaluate cell death? Iron uptake would be trackable much quicker.
• The legend for 2F is awkward (BSADQRED)
• Why are WT cells not included in Fig 2G?
• The biochemical characterization of NCOA4 in the LRRK2-arm cells is a great experiment and strength of the paper. The field would benefit by a bit further interrogation, other detergents, etc.
• Have the investigators looked for aberrant Rab trafficking to lysosomes in the LRRK2-arm cells? Is pRab8 mislocalized compared to WT? Other pRabs?
• The expression levels and therefore stability of the ARM fragment is not shown. This is necessary for interpretation. While very intriguing, the data in Aim 3 rely on the assumption that the ARM fragment is expressed, and at comparable levels to G2019S to account for phenotypes. The generation of second clone is admirable, but the expression of the protein must be characterized. This is especially true because of the different LRRK2 levels between WT and G2019S. One could easily conceive of exogenous expression of a tagged-ARM fragment into LRRK2 KO cells, for example, as another proof-of-concept experiment. If it is truly dominant, does this effect require or benefit from some FL LRRK2? It seems easy enough to express the LRRK2-ARM in at least WT and KO RAW cells.
• Does iron overload induce Rab8a phosphorylation in a LRRK2 KO cell? This would be a solid extension on the ARM data and support the important finding that an additional kinase(s) can phosphorylate Rab8a under these conditions, and while not unexpected, this may not have been demonstrated by others as clearly. It also addresses whether the ARM domain is important to this other putative kinase(s), which may add value to the authors' model.

Minor concern - the abstract but not the introduction emphasizes a hypothesis that loss of neuromelanin may promote cell loss in PD (through loss of iron chelation), while post mortem studies are by definition only correlative, early works suggested that the higher melanized DA neurons were preferentially lost when compared to poorly melanized neurons in PD. This speculation in the abstract is not necessary to the novel findings of the paper.

Significance

This study could shed light on a both novel and unexpected behavior of the LRRK2 protein, and open new insights into how pathogenic mutations may affect the cell. While studied in one cell line known for unusually high LRRK2 expression levels, data in this cell type have been broadly applicable elsewhere. Give the link to Parkinson's disease, Rab-dependent trafficking, and iron homeostasis, the findings could have import and relevance to a rather broad audience.

Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

Learn more at Review Commons

Reply to the reviewers

We would like to thank all the reviewers for their comments and suggestions.

Please find below our point-by-point response to the Reviewers' comments, which details the corrections already made and outlines the planned revisions, experiments, and analyses.

Reviewer 1

Major comments:

• Reviewer 1 commented that the 'manuscript would greatly benefit from having someone spend time on the figures, and associated text, to ensure they are fully comprehensible'. We agree wholeheartedly with the reviewer and apologise. We have now revisited the text, figures, and associated figure legends to ensure that they are more easily accessible and fully comprehensible to readers from across disciplines. This includes adding labels to point out specific anatomical features on images, and ensuring figures and text align. Further specific examples are included in the points below.
• In response to concerns raised by Reviewer 1 relating to: Figure 1 and the lack of figure citations; 'the persistence of mCherry in the H2B Fucci'; how mCherry seems to persist longer in H1 (compare Figs 1D and 1G)':
• We apologise for the lack of figure citations in the text. We have now reworked the figures relating to the constructs (original Figures 1 and S1) and have made these Figures 1, 2 and S1 in our updated version.
• Figure 1 is now an introductory background figure which illustrates the differences between Fucci(SA) and Fucci(CA) reporters, with additional details provided in the associated legend, and call outs to the figure starting in the introduction.
• Regarding 'the persistence of mCherry in the H2B Fucci', what we are trying to articulate is that the mCherry degradation that we observed in the Fucci(2A) expressing DF1 cells extended beyond the end of S phase and into G2/M, compared with what would be expected (Revised Figure 2H, arrows).
• We have now replaced these montages with a more representative example. Additionally, the new images (Figures 2C and 2G) are synchronised (both starting at G2/M), restricted to a single cell cycle, are larger in size, and have the cell cycle stage labelled. We believe these changes will aid interpretation.
• Specifically relating to the lack of labelling in Figure 3A, we agree that this figure was not labelled sufficiently, and neither was there enough detail included in the text or figure legend for readers to follow easily and make their own conclusions. We have now added additional labels to this figure, broken the figure down into more panels (Figures 4A-4D in revised manuscript), and included more detailed descriptions in the associated figure legend and text.
• We thank the reviewer for making the important point that it is 'hard to know where the biosensor is reporting patterns that are already well established (eg neural tube), and where the biosensor is reporting patterns that are novel - and if so, what these patterns are' which was made more challenging by insufficient references to previous studies.
• Firstly, as for the point above, we have now added labels to many of the panels (Figure 4 in revision), including highlighting features such as the non-proliferative dermal condensates and demarcating the proliferative retinal pigmented epithelium (Figures 4F and 4G in revision). Secondly, we have also now included additional references in the text, specifically relating to the neural tube, digits, and forming feathers, where our proliferation profiles are consistent with previous literature.
• With regards to the Reviewer's comment regarding the difficulty in drawing conclusions 'about cell cycle in different tissue layers without sectioning' in original Figure 3B we will include more sections of FuChi embryos which include structures such as mesenchymal condensates.
• To make our data on cell cycle stages as 'cells egress from the primitive streak, to form prechordal plate' clearer we have added additional labels to the figures (Figures 4B and 6E in revised manuscript). We will complement this adding sections of gastrulating FuChi embryos to further demonstrate the cell cycle status of cells that form the pre-chordal plates.

Minor comments

• We have added additional references relating to the data in original Figure 3 (now Figure 4 see above), and any new descriptions of known proliferation profiles that we include will have appropriate citations.
• In this current revision we have addressed figure call out issues, and added labels to enhance readability, clarity and data interpretation. Reviewer 2

Major comments

• Reviewer 2 rightly pointed out that the 'description of the bicistronic tandem-Fucci(CA) system in paragraph 6 is not consistent with what is described in the original bibliographic reference indicated by the authors'. We have now added additional text to properly explain the CDT1 probe dynamics, as per the cited manuscript, and also referenced the schematics to help readers.
• To address whether the FuChi model can be accurately 'used to study embryogenesis' and following up on the suggestion to 'indicate if the size of the embryos is comparable to the wildtype' we have now included size comparisons of FuChi and wild-type/non-transgenic embryos at mid (E9) and late (E18) gestational stages demonstrating that there is no significant difference between genotypes during embryogenesis (Figure 3D in revised manuscript). For all earlier stages, we did not see any developmental or size differences. We believe if there were any differences, these would be reflected in size at the mid and late gestational stages we analysed.
• Reviewer 2 made very valuable observations and suggestions regarding our data and interpretation of somitogenesis, specifically in response to our sentence saying that "the mesenchyme, which is predominantly in G1 as they undergo condensation". Furthermore, they noted that Supplementary Video 4 "shows distinct green fluorescence (S) in the presomitic mesoderm for the first hour or so, only then turning to magenta (G1)". We were asked to review the sentence/video to clarify if this is a significant finding or if this is not representative of their observations.
• We thank the reviewer for this suggestion. From looking again at our timelapse movies, and also analysing additional static images, we agree that presomitic mesoderm (PSM) does appear to be green (S phase), which then may transition to G1 as the somites form. To address this, we plan to quantify cell cycle status in the PSM on embryos to see if this is a significant finding.
• We hope this quantification of the PSM may also enable us to include discussion on how our findings relate to the Cell Cycle model for somitogenesis proposed in the Collier et al, 2000 paper suggested by the Reviewer.
• We agree with the Reviewer that "the fluorescence profiles in original Figure 4C do not seem similar regarding the Myc-tag epitope" and believe this difference is likely just a reflection of the part of the image we used. We will include a more representative image once we have repeated the staining.
• Reviewer 2 has asked for quantitative support for our fluorescence-based interpretations. We thank the reviewer for this suggestion and are now planning to perform quantitative analyses of different tissues (similar to our quantification in germ cells) and in embryos to support our observations. These will include the PSM (see above), neural tube, intestine, and early embryos (also see Reviewer 3 response for blastoderm quantification).
• Since our original submission, we have further refined our in situ hybridisation protocol on FuChi embryos (Figures 5A & B in revision), finding that strong reporter expression is maintained for all the fluorescent proteins of the H1-Fucci(CA)2 reporter. Therefore, the "notably fainter" appearance of the hGMNN-mVenus in Figure 4A from the first version of the paper was likely a result of the experimental protocol not being 100% optimal.
• *

Minor comments

• We have reordered the paragraphs relating to the different Fucci versions in the introduction as per the suggestions by the reviewer for better clarity.
• To address the issues with Fucci system nomenclatures which made reading difficult, we have now added a background figure (new Figure 1 in revised draft) which is cited in the introduction, made sure constructs are introduced appropriately, and ensured we are consistent with our nomenclature.
• Supplementary Figure lettering corrected.
• All figure panels are now mentioned in the main text, and the incorrect call outs noted by the Reviewer have been corrected
• Removed period and included clarifying statement in the figure legend relating to the comment regarding the extraembryonic region in Figure 5 (original) / Figure 6 (revised).
• Other issues raised relating to reference duplication and missing words have been resolved.
• We have corrected the legend of Figure 1 of the original paper, see related Reviewer 1 response provided above.

Reviewer #3

Minor comments

• We have corrected all the figure call outs (see responses to similar comments by Reviewers 1 and 2) to ensure that all data presented is accurately reported.
• We would like to thank the reviewer for suggesting modifications to the cell cycle montages (original figures 1D, 1G and 2F). We agree it would help the reader to enlarge the image, and therefore reduced the montage to include just one cell cycle, and have also included annotations of cell cycle stages in Figures 2C and 2G of the revised manuscript. We have also added some labels to Figure 3E (original figure 2F) and enlarged this.
• In response to Reviewer 3's comment regarding fluorescent intensity. We quantified fluorescence levels in multiple individual DF1 cells expressing either the H1.0-Fucci(CA)2 or H2B-Fucci(SA)2 reporters, and this is shown as the fluorescent index in Figures 2D, 2E, 2H and 2I of the revised manuscript, where reporter levels were measured across time. In terms of overall mean intensity levels of the reporters, we found the reporters to be comparable in brightness and have similar mean intensity levels across the cell populations in the flow cytometry data (Figures 2F and 2J).
• To enhance speedy interpretation, we will also process our supplementary videos to include annotations and arrows to highlight key cells and events (e.g. a cell undergoing mitosis).
• As recommended by Reviewer 3, we have now quantified cell cycle status in blastoderm cells, confirming that a high proportion are in the G2/M phase. We will include these data in the final revision, which will complement our planned quantification of cell cycle status in other tissues (see response to Reviewer 2).
• For our final revision, we will include higher magnification/zoomed in images of selected regions of the somites, neural tube (lumen) and retina (epithelium). Revisiting our images of the neural tube showed that dividing cells lumen did so in the perpendicular plane and we will include these images in our revision to provide further evidence of the fidelity of the FuChi reporter. We thank the reviewer for this excellent idea to show the efficacy of our system.
• To address the levels of proliferation in somites, we plan to generate a cropped video with a fixed ROI to enable proliferation in individual cells of the forming somites to be more readily visualised. This will be further complemented by the quantification of cell cycle status in forming somites (see responses to other reviewers).
• We have added lines to the discussion regarding the use of our reporter in other conventional model systems.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

Referee #3

Evidence, reproducibility and clarity

The manuscript by Sudderick and colleagues describes the development and characterisation of a new generation of cell cycle reporter that can distinguish between cells in G1, S, G2 and M phases. Furthermore, the authors have developed a transgenic chicken line incorporating this reporter and demonstrated faithful discrimination of cell cycle stages in the in vivo context of developing transgenic embryos. Of note is the addition of epitope tags, which facilitate discrimination of cell cycle stages in tissue fixed using various techniques. This is a very important paper for the following reasons:

• The authors have achieved faithful discrimination of all four cell cycle stages, which is a major advance in itself.
• This generation of the FuChi transgenic chick is of enormous importance. This will facilitate accurate in vivo studies in a broad range of fixed and living tissue types and is a major milestone in the further establishment of the chick as a transgenic model system.

Th characterisation of the cell cycle reporter as presented is robust and convincing. The authors further demonstrate the potential utility of the FuChi chickens through their observation of partial cell cycle synchrony during onset of development. I therefore only have minor suggestions that may facilitate easier interpretation of their data.

Results 2

• I can't see any mention of Figures 1C and D. Presumably the authors have carried out fluorescence intensity measurements using the two cell cycle reporters here, but this is not mentioned in the main text.
• Figure 1D&G: I find these difficult to follow given the small size of the cells as presented. The authors may consider enlarging these and clearly annotating for cell cycle stage. They may find it helpful to focus on a single cell cycle, although I appreciate that displaying two cell cycles strengthens the claim of efficacy of the newly developed sensor. The supplementary videos associated with these figure panels are excellent as they display several cells with faithful reporter activity, but again, the authors may wish to annotate a few of these cells to enhance speedy interpretation. I have similar comments for Figure 2F and the associated movie.

Results 4

• The authors state that a large proportion of blastoderm cells were in G2/M. They may wish to formally quantify this, perhaps by performing simple cell counts in designated regions of interest. A similar quantification for gastrulating embryos would also be helpful.
• It would be helpful to see zoomed in images of selected regions of the somites, neural tube and retina displayed in Figure 3B. This would be particularly appropriate in the context of the neural tube and retina (which are not discussed in the main text) as the positioning of the nucleus is defined by the stage of the cell cycle and should therefore serve to highlight the efficacy of the reporter.
• Video 4 beautifully demonstrates the high levels of proliferation in somites, but again, it would be useful to have a zoomed in view. I appreciate the difficulty involved in doing this, given the movement of the embryo, but perhaps the authors could focus on a fixed ROI or present a separate movie of a few cells undergoing a full cell cycle.

Discussion

• The authors could perhaps expand on their discussion about potential utility in other conventional model systems (e.g. mouse, fish, etc).

Significance

General assessment: A timely piece of work that introduces a faithful cell cycle reporter that will be of broad interest.

Advance: The ability to discriminate between all four stages of the cell cycle is a clear advance here.

Audience: Broad interest, including those studying cell cycle and embryonic development in several tissue contexts.

Expertise: Chick embryology, in vivo live imaging, neurogenesis, cellular developmental biology

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

Referee #2

Evidence, reproducibility and clarity

Summary:

This work presents a novel transgenic chicken model with fluorescent reporters that allow in vivo monitoring of the four phases of the cell cycle. To achieve this, the authors clearly identify the limitations of previous Fucci systems and developed an optimised reporter construct that overcomes the major technical challenges identified. Addition of epitope tags to cell cycle stage-specific markers further enables antibody detection in fixed tissues. Proof of concept is provided by live imaging of chick embryos in early developmental stages, evidencing dynamic cell cycle states in tissues and migrating cells.

Major comments:

1. Introduction: Description of the bicistronic tandem-Fucci(CA) system in paragraph 6 is not consistent with what is described in the original bibliographic reference indicated by the authors. Namely: "...accumulation of the CTD1 probe..." should be expected in the G1-S transition (not S-G2) and the yellow reporter should be expected in G2 and M phases (not S and G2, as described). Please review this portion of the text.
2. The authors state that "Of note, hatched FuChi chicks are initially smaller than wild type counterparts but grow at comparative rates and are fertile". If the model is to be used to study embryogenesis, it would be useful to indicate if the size of the embryos is comparable to the wildtype, at least for the major developmental stages mentioned in the manuscript.
3. When referring to somitogenesis, the authors state "...the mesenchyme, which is predominantly in G1 as they undergo condensation". Suppl Video 4, however, shows distinct green fluorescence (S) in the presomitic mesoderm for the first hour or so, only then turning to magenta (G1). The authors should review the sentence/video to clarify if this is a significant finding or if this is not representative of their observations.
4. (Optional) It would be interesting to describe if the authors' observations of cell cycle dynamics in the presomitic mesoderm support the proposed Cell Cycle model for somitogenesis (Collier et al., J.Theor.Biol.2000).
5. The fluorescence profiles in Figure 4C do not seem similar regarding the Myc-tag epitope (contrarily to what is stated). The authors should rephrase or revisit this image to clarify their findings.
6. Quantitative support for several fluorescence-based interpretations made throughout the manuscript. In some instances, conclusions are drawn from qualitative differences in signal intensity. For example, the statement in Fig. 4A that hGMNN-mVenus appears "notably fainter" than the other reporters. Incorporating simple quantitative analyses would strengthen these claims and ensure that observed differences reflect biological behaviour rather than technical or optical factors.

Minor comments:

1. Organization of the information in the Introduction: Paragraphs 3-5 introduce sequentially improved versions of the Fucci system. Then, paragraph 6 returns to the system described in the 4th paragraph. Authors should consider including paragraph 5 (description of Fucci4 and its limitations) just prior to the description of chickens as valuable developmental models (current paragraph 8) for clarity of the text.
2. Fucci system nomenclature. Many different Fucci systems are mentioned, but nomenclature consistency throughout the manuscript is lacking, which makes reading difficult. For example, the terms "Fucci(SA)2" and "Fucci(CA)2" should be defined in the introduction, as they are employed to describe the construction of the new biosensor in the following sections.
3. Some figure panels are not mentioned in the main text (for ex. Figures 1B and C, Figure 2C)
4. The legend of Figure 1 (D & G) mentions "denoted by *", but the * seems to be missing in the figure.
5. Supplementary Figure 1 has two D panels (and is missing the E).
6. In the main text, where it reads "...Flow cytometry analysis of three independent PGC lines... (Figures 2G & S2E)", S2E should be replaced by S1E.
7. In the Figure 4A legend, hCDT1-mVenus should be corrected to hCDT1-mcherry. Also, it is not clear why the authors state that "hGMNN-mVenus expression is notably fainter compared with hCDT1-mVenus and H1.0-mCerulean expression".
8. In Figure 5E, the optical sections "i" seem to pertain to the extraembryonic tissue/area opaca and not to anterior mesoderm, as stated in the figure legend. Also, there is a period between "prechordal plate" and "and" in the legend's last sentence.
9. Discussion: The last sentence of the third paragraph lacks "to" between "used" and "interrogate".
10. References 10 and 23 are identical.

Referee cross-commenting

I agree with all comments from reviewers 1 and 3

Significance

This is a beautiful paper, describing a long sought-after model system to study cell cycle dynamics in vivo. The methodological details are thorough, and the results obtained are clearly presented, highlighting the utility of the new model in various embryonic stages and tissues/organs.

This work is of pivotal importance to the developmental/stem cell biology community, as well as to the wider community that employs the chicken embryo as a preclinical model to assess therapeutic or teratogenic potential of biologically- or chemically-derived products.

My expertise is in chicken embryo development, namely gastrulation, somitogenesis and limb bud outgrowth.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

Referee #1

Evidence, reproducibility and clarity

Summary:

The manuscript reports the development of a novel Fucci (Fluorescent Ubiquitination-based cell cycle indicator) system for analysing cell cycle analysis, including live imaging of cell cycle. The novel biosensor (H1.0-Fucci(CA)2) has been developed for analyses of chick cells and tissues: chick embryos are a valuable developmental model that have (and in the future, will) particularly informed our understanding of early stages of embryogenesis, and of development of numerous tissues, including the neural tube, somites, limb bud. The authors conclude that the novel system has advantages over previous Fucci systems, including faithful labelling of all four cell cycle phases. Importantly, the authors have generated a stable germline of H1.0-Fucci(CA)2 transgenic chicks, enabling, for the first time, the discrimination and tracking of cells in all 4 phases of the cell cycle - i.e. in vivo studies of cell cycle progression in vivo, in intact tissues and organs. Additional epitope tags mean that the biosensor can be detected in fixed tissues, enabling comparison of cell cycle with expression of mRNA and proteins that mediate other aspects of development/label particular cells and tissues. The authors map proliferation dynamics across numerous tissues in the developing chick, at numerous stages of development, and conclude in particular that transition from S phase may be a key morphogenetic event in gastrulation, as mesendoderm cells leave the primitive streak to form embryonic stuctures such as prechordal plate

Major comments:

The novel biosensor looks to be an incredibly useful tool, and the manuscript suggests patterns of cell cycle progression in different tissues, and at different points in time, that look intriguing. But it is sometimes difficult to draw the strong conclusions suggested by the authors because the text and figures are sometimes difficult to follow. The manuscript would greatly benefit from having someone spend time on the figures, and associated text, to ensure they are fully comprehensible.

Specifically:

Conclusion1: That the new FUCCI biosensor is a superior cell cycle probe, better at discriminating all cell cycle phases than previous versions. I was very convinced by the vidoes (video 1 and 2) but had problems with Figure 1. Potentially, this is because I am not an expert in these types of analyses - but it was not helped by the fact that components of the figure were not cited in the text. I was particularly confused by the statement remarking on 'the persistence of mCherry in the H2B Fucci' as mCherry seems to persist longer in H1 (compare Figs 1D and 1G). Please explain, in the Figure legend, why this appears to be the case.

Conclusion 2: that the FuChi chicks are the first viable stably expressing avian cell cycle biosensor model. I agree, and the authors should be congratulated on the development of this important tool.

Conclusion 3: the authors monitor cell cycle progression in chicks, in vivo, looking at stages from blastoderm, through gastrulation, and into organogenesis, and draw various conclusions

For example: Fig 3A and text: 'as gastrulation progresses, the primitive streak an presomitic mesoderm display...., whereas the .... And neural plate contains...'

Figure 3A covers an enormous range of stages and tissues. The figure is barely labelled. The text and figure need to better align, and key features in each figure panel need to be labelled so that the reader can better follow, and draw conclusions.

Fig 3B: Reports expression in numerous tissues. There are some beautiful examples of cells segregating relative to cell cycle - for instance, in the neural tube. But I found it hard to know where the biosensor is reporting patterns that are already well established (eg neural tube), and where the biosensor is reporting patterns that are novel - and if so, what these patterns are. Again, this is not described adequately in the text (for instance, there is no mention of the neural tube). And in some cases, references are provided (allowing comparison with previous studies) - but in other cases, there are no references to previous studies. The reader must be given the opportunity to compare this study with previous studies.

Overall - I can appreciate that there are some fascinating patterns, but it is very difficult to draw the conclusions suggested by the authors. Primarily this is due to poor labelling of figures, and lack of clarity between figures and text, and poor referencing. Additionally, it is not clear that strong conclusions can be drawn about cell cycle in different tissue layers without sectioning some embryos.

Fig 3C: The authors remark 'The results confirm that the ... FuChi embryos recapitulate known cell cycle profiles of those tissues'. See my comments in 3B.

Conclusion 4: Robust stability of biosensor in fixed tissues. I agree, and the authors should be congratulated for having made a construct that can be paired with in situ hybridisation and immunohistochemistry - this is invaluable.

Conclusion 5: The authors investigate the potential of the new system for live imaging, and focus on a couple of novel dynamic examples.

The data indicating that PGCs at initial migratory stages are not undergoing frequent cell division is clear.

However, the data indicating that cell cycle status changes as cells egress form the primitive streak, to form prechordal plate, is not clear. The figures need to be better labelled, and the text needs to be more clear (eg ' and prechordal plate. and anterior mesoderm'..

Minor comments:

• Specific experimental issues that are easily addressable.

I would recommend that the authors section some embryos, to better support key conclusions (eg in figure 3 and 5) - Are prior studies referenced appropriately?

Not always - see comment above (Fig 3) - Are the text and figures clear and accurate?

No - this needs work. Not all figures cited in text, or cited in wrong order; Figures are poorly labelled - making it hard to follow - Do you have suggestions that would help the authors improve the presentation of their data and conclusions?

Label figures more carefully and ensure figures and text align

Referee cross-commenting

I agree with all comments from reviewers 2 and 3

Significance

• Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.

Technically this is a fantastic resource. As detailed above, the novel biosensor (H1.0-Fucci(CA)2) has been developed for analyses of chick cells and tissues: chick embryos are a valuable developmental model that have (and in the future, will) particularly informed our understanding of early stages of embryogenesis, and of development of numerous tissues, including the neural tube, somites, limb bud. Increasingly, studies show the importance of cell cycle for development, differentiation and morphogenesis - it is a huge breakthrough to be able to perform in vivo studies of cell cycle progression in intact tissues and organs.<br /> - State what audience might be interested in and influenced by the reported findings.

Broad basic research, including developmental biologists, stem cell biologists, modellers. - Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.

Developmental biologist, with expertise in chick

There should be an option for this!

In the meantime, a workaround: How to get a serial comma: Use en or en-US instead of en-GB.

new Intl.ListFormat("en-US", { style: "long", type: "conjunction" })

It is always valuable to give the option to developers to use it how they want. The developer may want to use it without necessarily making it a user-controllable UI preference!!

Me gusta en un 50/50 esta forma de pensar, si bien nada es perfecto y todo puede mejorar y evolucionar con el tiempo, no me cerraría a pensar de que estoy lejos o que no podría llegar a la meta que me propuse en cierto momento. Todo en la vida es resiliencia y mejora continua de procesos de manera progresiva y alcanzable.

Me parece algo totalmente genial (dejando de lado de que no todo el mundo tiene un récord guinness y todos los requisitos que solicitan para tenerlo) sino por el hecho de que no es algo aleatorio.

Recuerdo haber leído sobre ese récord hace un tiempo y si no me equivoco era sobre recorrer cierta cantidad de estaciones de metro (unas 400 y algo) en un tiempo de 24 horas ("pasaditas") y que estuvo vigente hasta cierto tiempo, lo que me parece fascinante es que cómo dije anteriormente, no fue algo esporádico, fue algo planeado, algo planificado en equipo

Cómo toda gran idea novedosa o innovadora que nace de la incomodidad ...

Ser disruptivo y crear algo que cambie y mejore las reglas convencionales es algo que siempre he de admirar. Tener la convicción de diseñar algo que se sabe que reúne lo mejor de varios sistemas es algo que no todo el mundo hace, si bien quisieron hacer algo más "pequeño, propio y privado" (que se entiende muy bien, no por el tema de envidia o privatización sino porque quizá uno cómo persona no dimensiona el impacto de sus creaciones), algo que me llamó la atención es que fueron de lleno a crear algo a la altura de los lenguajes de alto nivel (básicamente que se pueden hacer más y mejores cosas sin tantas líneas de código), ósea que simplemente no fue un típico proyecto que ya existía, sino que intentaron ir más allá de una vez, simplemente adelantados a su tiempo, es increíble

I think the people have forgotten what social media was created for - a means to connect people around the world. Forgetting this core idea is probably what has made social media so harmful as now it is used for almost every aspect of our life except the simplicity of connecting with someone.

I think this really highlights the difficulty we're facing with social media these days as while we all know the toxic aspects of it we cannot get it our of lives as in truth it's not as toxic if used in moderation. However due to it's addictive nature most people cannot use it in moderation and therefore it only adds harm to people's life.

This quote shows why it’s so hard to address most issues around social media. It’s almost a necessary evil, as it is an essential part of all of our lives. It is difficult to keep up with real life if you’re not connected online too, so solutions to social media addiction or negative effects must be found on the other end — the developers’ end.

I like how this compares social media to reading a book, because it shows the effect isn’t automatically negative. But the difference is that platforms are designed to keep extending that state indefinitely. It feels less like getting absorbed in something and more like getting stuck.

This highlights how “digital detox” can be helpful as a personal tool, but treating the internet as inherently toxic turns into the same kind of nostalgia we’ve seen before. Collee’s point is that idealizing a return to “offline” can ignore who is actually included or excluded in that fantasy, similar to how “wilderness” was romanticized while Indigenous people were erased.

This is such a real issue—when edited photos become the “normal” standard, people can start feeling like their unfiltered face or body isn’t good enough. Cosmetic surgeons are noticing that some patients now bring filtered selfies as goals, which shows how social media can reshape self-image in unhealthy ways. It’s a good reminder that what we see online is often curated or altered, not a fair baseline for how real people should look.

GENDER, SEXUALITY, AND SHIFTING STYLES: KNOWLEDGE PRODUCTION AND CODIFYING LANGUAGE USE IN STYLE GUIDES

CLEAVAGE THEORY ORIGINALLY says that national politics is representation not of party organisation, but of "UNDERLYING SOCIAL CONFLICTS" and therefore input from electorate.

Seems remarkably democratic in that electorate are ultimately the ones who are steering the electoral ship / [arty platform directions.

PAST examples of cleavages (which, again, in this context simply means disruption or shock) include industrialisatioon and the urban/rural divide -> nationalism vs obligations to the supranational Church, etc. -> In other words, the cleavage NOW is between supranational supporters (EU) and NATIONAL supporters reacting to perceived encroachments on EU identity.

TO CONCLUDE -> authors aregue that transnational cleavage is EXPLOITED BY FAR RIGHT PARTIES BECAUSE THEY BECOME SINGLE ISSUE AND FOCUS ON THESE ISSUES VERY KEY TO VOTERS, while mainstream parties are not primarily devoted to immigration, etc -> are at a disadvantage -> like that thesis that says that actors in referendums who CONTROL THE NARRATIVE of referendums have the advantage -> can make it about a single issue, etc.

"Radical right parties take more extreme positions on these issues, place more salience on them, and exhibit greater internal unity than mainstream parties. By virtue of their commitment to GAL values, green parties are located at the alter-pole. Just as the religious cleavage and the class cleavage were raised by Catholic and socialist parties on one side of the divide, so the transnational cleavage is mobilized by radical right parties at one extreme. As the transnational divide has become salient, mainstream parties have been compelled to compete on issues that lie far from their programmatic core."

Whole idea of cleavage theory is that parties and their platforms respond to CRISES and a shift in voter attitude as opposed to any internal decision making process. THEREFORE -> policy shifts by parties are SUDDEN as opposed to INCREMENTAL

"They suggest that party systems are subject to discontinuities rather than to incremental change, and that the response of a party system to exogenous change comes from voters rather than parties."

Again, all of this IS REACTION TO immigrqtion, increased supranational govt / control by "foreigners," Eurozone, etc. All about loss of sovereignty basically.

"as a reaction to reforms that have weakened national sovereignty, promoted international economic exchange, increased immigration and exacerbated cultural and economic insecurity."

TRANSNATIONAL CLEAVAGE: - i.e., the cleavage is the term for the DISRUPTOR / SHOCK - THIS shock is transnational -> i.e., EROSION OF NATION STATE SOVEREIGNTY (This is the shock) - Penetration of nation state politics by "external actors" (immigratants, "foreign" governments, etc.)

OKAY -> so it is EU project as a whole that is a "shock" to the elecoral system (comparable to previous crises outlined in 1967 article, unclear what those are) BECAUSE it strips nations of absolute sovereignty and hands some of that sovereignty to "foreigners" (i.e., other member state govts) ALSO facilitates immigration -> seen as competition w/ jobs and housing BY electorate.

"The European Union (EU) is itself such a shock, because it introduces rule by those who are regarded as foreigners, diminishes the authority exercised by national states over their own populations, produces economic insecurity among those who lack mobile assets, and facilitates immigration. Immigration is perceived as a particular threat by those who resent cultural intermixing and the erosion of national values, by those who must compete with immigrants for housing and jobs, and, more generally, by those who seek cultural or economic shelter in the rights of citizenship."

Es bastante bueno, diferencial e innovador que se permita crear y dirigir la creación de páginas web de acuerdo a lo que desean y quieren las personas. Mientras que otros servicios se encargan de ser generadores netamente de sitios web empresariales o de market, esta permite dirigir su servicio a otro del mercado, cómo el de los videojuegos

Si bien esto puede ser un ganchazo comercial muy bueno, es una razón valida para optar por herramientas gratuitas y propias que de verdad se preocupen y sean garantes de la protección de los datos, datos personales y contenidos de las páginas web que creen las personas

Si bien comparten ciertas características cómo algunos referentes que son lideres en la generación rápida de sitios web cómo GoDaddy o Wix, me llamó la atención que sean laxos en cuanto a funciones, contenidos e infraestructura, mientras que otros sitios que de cara ya son de pago o restringen el uso de cierto tipo de contenido, esta promesa que compite contra estos lideres potencia y libera la capacidad creativa de los usuarios de forma gratuita, propia e intuitiva

NOTE DE SYNTHÈSE : Dans la tête d'un colérique

Introduction

Cette note de synthèse explore les facettes de la colère présentées dans les extraits de l'émission "Dans la tête d'un colérique".

Le documentaire examine la nature de cette émotion souvent perçue négativement, sa gestion individuelle et collective, ses manifestations, ses fonctions insoupçonnées, et les conséquences de son refoulement ou de son expression violente.

À travers des témoignages, des analyses de spécialistes et des expériences, l'émission offre un éclairage nuancé sur une émotion complexe et puissante.

Thèmes Principaux

La perception sociale de la colère et son contrôle: La colère est largement considérée comme une émotion négative et "mal vue".

L'éducation et les règles de bienséance nous incitent à la contrôler.

La fonction intrinsèque et l'utilité de la colère: Malgré sa mauvaise réputation, les spécialistes affirment que la colère est nécessaire.

Elle peut nous protéger et, de manière surprenante, augmenter considérablement nos performances physiques et cognitives.

Les manifestations et mécanismes de la colère individuelle:

Le témoignage d'Eduardo illustre comment la colère peut être déclenchée par la peur (face à une agression) ou la frustration (avec ses enfants), entraînant des réactions physiques intenses ("tout ton corps qui change si ce mr bouillir... les yeux qui devient pour les rouges tout ça").

Chez un colérique, la tension monte vite et très haut, rendant difficile la prise en compte d'autres perspectives ("il est plus capable de penser... il n'ya plus que son point de vue qui compte").

La colère dans les relations proches:

Les émotions, y compris la colère, débordent davantage avec ceux qu'on aime.

L'anticipation des réactions de l'autre ("je savais que tu allais faire ça") et le système d'attachement (sécurisé ou insécure) influencent la gestion de la colère dans les relations intimes.

Un système d'attachement insécure peut amplifier les émotions négatives ("ça va alimenter active est encore plus mes émotions de tristesse de colère").

La réaction face à la colère d'autrui:

Être confronté à la colère d'un inconnu provoque souvent la "pétrification" ou la "sidération".

La colère fait peur car elle "menace potentiellement l'intégrité d'autrui", mais aussi "l'intégrité des règles de civilité".

On ne sait jamais quelles sont les "limites" de la personne en colère, d'où la peur et le retrait.

Les conséquences de la colère non maîtrisée et violente:

La colère, surtout lorsqu'elle devient violente, fait souffrir non seulement les proches mais aussi les personnes colériques elles-mêmes.

Dans les cas extrêmes, elle peut empêcher de vivre et mener à des comportements autodestructeurs ou hétéro-agressifs.

Le témoignage de Mischa, une femme violente, met en lumière la honte et le caractère dévastateur de sa colère ("c'est très violent c'est c'est comme un tremblement de terre").

La gestion et la canalisation de la colère:

Refouler la colère est comparé à un "cancer" qui peut créer des "pathologies physiques".

Apprendre à réguler sa colère ne signifie pas l'éradiquer, mais trouver d'autres stratégies pour soulager la tension interne.

L'utilité fonctionnelle de la colère: Communication et positionnement:

La colère est un "outil de communication" dès l'enfance et permet, à l'âge adulte, de "mettre des limites" et de ne pas se faire "bouffer par les autres".

Performance physique: Une expérience a démontré que la colère (induite par un sentiment d'injustice) peut augmenter la force physique ("augmentent leur force physique").

La colère agit comme un "moteur", un "booster" qui "permet d'optimiser les performances" physiques car elle est associée à un niveau d'"activation" et d'"éveil" élevé.

Performance cognitive (inconsciente):

Une autre expérience suggère que l'activation inconsciente du concept de colère (par des images subliminales) peut faciliter les performances cognitives et rendre le cœur plus efficace ("économise son énergie et devient plus efficace").

Ce processus doit être inconscient pour fonctionner.

Performance sociale et politique: La colère peut être utilisée consciemment pour "faire passer un message", comme le fait l'entraîneur Bernard Challandes.

La "colère sociale" est perçue comme un "moteur" essentiel pour "changer les choses" et maintenir l'engagement dans les mouvements collectifs (comme la grève du climat).

Les colères collectives ont historiquement fait peur car elles sont "synonyme d'émeutes" et de "révolution", mobilisant les individus en une force collective qui peut ébranler l'ordre établi.

Le traitement de la colère:

L'approche pour gérer la colère dépend de sa nature.

La psychiatrie peut être pertinente si la difficulté à contrôler la colère est constante, survient dans différents contextes et est associée à des comportements délétères, suggérant un trouble psychiatrique sous-jacent (comme une dépression).

Cependant, pour des difficultés comportementales, des approches se concentrant sur l'apprentissage de la gestion des émotions et le renforcement de l'estime de soi sont également efficaces, notamment pour les femmes violentes qui recherchent souvent de l'aide comportementale plutôt que psychiatrique.

Idées ou Faits Importants et Citations Clés

La colère, une émotion nécessaire:

"pourtant les spécialistes l'affirment nous ne pourrions pas vivre sans colère elle nous protège et vous le verrez dans cette émission elle augmente considérablement nos performances physiques et cognitives".

La montée rapide de la colère chez certains:

"[chez Eduardo] la tension monte très vite un très rapidement mais aussi très haut et qu'à ce moment là sur une émotion qui semble être de la colère il est plus capable de penser".

La colère dans les relations proches:

"les psychologues le disent les émotions déborde davantage avec ceux qu'on aime".

L'influence du système d'attachement:

Un système d'attachement insécure "va très vite interpréter les signaux que me donne l'autre comme il est en train de me laisser tomber... ça va alimenter active est encore plus mes émotions de tristesse de colère ou tout autre émotion négative".

La peur face à la colère d'autrui:

"les émotions de colère elles elles font peur parce que menace potentiellement l'intégrité d'autrui... face à la colère d'autrui surtout des personnes inconnues on est plutôt dans un état de pétrification de sidération".

Les conséquences du refoulement:

"je pense que la colère c'est comme un cancer c'est que si on la garde à l'intérieur qu'on laval caen laval caen laval... réellement je pense que oui ça rend malade sa c'est sûr ça j'en suis sûre et certaine".

La fonction de communication de la colère:

"c'est un outil de communication déjà depuis la petite la prime enfance c'est un outil qui me sera utile aussi plus tard à l'âge adulte pour me positionner dans la vie mettre des limites... Si je me mets jamais en colère je me fais bouffer par les autres".

La colère comme moteur de performance physique:

"la colère c'est une sorte de moteur un booster qui va permettre d'optimiser les performances mais certaines performances performance physique".

La colère et la performance cognitive inconsciente:

"cette activation d'une idée de la colère de penser à la colère peut influencer comment je m'applique pendant une tâche... la colère peut faciliter nos actions". "tout ça doit être inconscient".

La colère collective comme moteur du changement social: "il faut une colère sociale pour changer les choses Sinon c'est quoi le moteur". "les colères collective elles ont toujours fait extrêmement peur parce que ça a été synonyme d' émeutes synonyme de l'annoncé de révolution".

Conclusion

L'émission "Dans la tête d'un colérique" démystifie la colère en la présentant non pas uniquement comme une émotion destructive, mais aussi comme une force intrinsèque et potentiellement utile.

Si sa manifestation violente ou constante peut avoir des conséquences dévastatrices pour l'individu et ses proches, la colère, lorsqu'elle est comprise et gérée de manière appropriée, peut servir d'outil de communication, de motivation et même de catalyseur pour le changement social.

Il est crucial d'apprendre à canaliser cette énergie plutôt que de la refouler ou de la laisser déborder de manière incontrôlée.

Version 3 of this preprint has been peer-reviewed and recommended by Peer Community in Microbiology.<br /> See the peer reviews and the recommendation.

term

a bar!

thats crazy

interesting

!!! important in a way that I cannot describe at the moment

heuristics for personal responsibiity

that's why I resonate with Hermetic principles

hermeticism is cosmic heuirtics

myth metaphor and mysticism

heuristic

dweb.archive

ipfs.indy0.net

Again a skill that can not be mastered. There will always be something new you learn each time you go to write something.

Reading this statement will help me give my daughter some grace in her classes. When it comes to her writing.

What I have gathered is that writing is a skill that can not be learned and when you think you have mastered it, You have to learn something knew. Its an UNMASTERABLE SKILL.

There is a certain tone and energy that every piece of writing has from a one page journal entry to the biggest award winning piece ever written, everything in-between. They all have their own structure, energy, setting , Their own VOICE!

This statement alone hits home with me. As i write a bio paper and the paper I am currently writing in this class are both very different writing styles. The papers i have written in other classes as well are all different than this one. The structure are all taught differently

SIDE NOTE: Not speaking about learning to write your A,B,C's: Even though there is meaning behind that as well.

Wardle makes a statement to emphasize: The meaning behind something that is written, can always change however there is always a particular reason its written

Este apartado tiene un poder y peso bastante grandes. El impacto de las redes sociales en las personas y en la industria es algo de doble filo, si bien se mencionan cosas tales cómo que "el impacto de la opinión pública puede extenderse de maneras inimaginables" y que las personas están interesadas en las nuevas formas en cómo se recibe la información, hay que tener en cuenta que esta misma información queda expuesta en la red (a veces hasta sin consentimiento ni conocimiento de las personas), Facebook y su creador se han vuelto inmersos en varias polémicas cómo ya sabemos, pero es este el riesgo de manejar datos sensibles, los algoritmos moldean a las personas, las redes sociales vuelven dependientes a estas personas, si bien la forma en cómo nos comunicamos y consumimos información ha crecido a pasos gigantes cabe recalcar que vamos perdiendo humanidad e identidad propia a base del consumismo provisto por la industria

Si bien, se define la comunicación de la información cómo algo "unilateral" -que está muy bien, dependiendo desde que arista se vea- cambiaría esto (Incluso con la mención "desde antes de la llegada del Internet") al hecho de que esto puede llegar a modificarse cómo algo BILATERAL en algunos o en la gran mayoría de casos.

Un caso puntual sería, donde un individuo difunde información y esta llega a un receptor o un espacio receptivo que está a la espera de este conocimiento para seguir difundiéndolo interactúa con este primer individuo y su conocimiento compartido creando y generando el famoso "intercambio de saberes".

Esto nace de que no es que haya un solo creador de conocimiento que simplemente se encarga de difundirlo y ya, sino que en su lugar, aparecería un agente externo que lo recibe e intercambia conocimiento con este

Me gusta cómo se hace un pequeño desglose (de la GRAN variedad de carreras vinculadas) sus características y funciones, ya que brinda una perspectiva llamativa sobre en donde y en qué se puede desempeñar un individuo, sin embargo, para hacer un poco más llamativo el apartado sobre las carreras, recomendaría asociar más de estas mismas

Si bien es una concepción general muy contundente y clara, desde un punto subjetivo no deja de ser susceptible a cambios y redefiniciones dependiendo de la persona y el campo desde el que se explique, ya que esto es un campo de acción tan amplio que no se puede limitar a una simple (o única) definición

Esto es algo completamente cierto que muchos de nosotros podemos llegar a olvidar o no tener en cuenta y es que se vive esa cooperación o integración de varios campos de acción, conocimiento o profesiones que permiten buscar soluciones más integrales en cooperativo

Si bien podría considerarse algo "básico" no puedo dejar pasar que es un bien necesario el hecho que es bueno eso de aclararle de forma inicial, a la audiencia y/o lectores que no hay que confundir o tergiversar conceptos por más parecidos o afines que sean.

Or i can?

I'm sure I shouldn't be able to do that.

hi devs?

also stated in the other article from this week "our story"

Doing this definitely makes it harder than It is truly is because we tend to look at it and think "Omg this is a lot" and we start getting overwhelmed by it.

This is very true we all take information in different ways and we all respond different to it due to us thinking and doing our work differently.

I think we all tend to do that from time to time and it does affect our quality of writing.

Rejet, victimisation par les pairs et émotions négatives : Synthèse des dynamiques d'influence en milieu scolaire

Synthèse opérationnelle

Ce document présente une analyse approfondie des recherches récentes menées par l'Institut universitaire Jeunes en difficulté concernant les liens entre l'isolement social, la victimisation par les pairs et les émotions négatives chez les élèves du primaire.

Les points saillants de cette étude sont les suivants :

• Prévalence élevée : Un nombre significatif de jeunes, particulièrement les filles, éprouvent une détresse émotionnelle quotidienne et un sentiment de non-acceptation dès le début du secondaire, des tendances amorcées au primaire.

• Renversement de la perspective traditionnelle :

Contrairement à l'idée reçue voulant que les problèmes relationnels causent les émotions négatives, les résultats indiquent que les émotions négatives (tristesse, désespoir) précèdent et prédisent souvent la victimisation.

• Boucle de rétroaction pour l'isolement : Il existe une relation bidirectionnelle entre l'isolement et les émotions négatives, créant un cycle d'aggravation mutuelle.

• Stabilité des traits vs États changeants : L'étude distingue les caractéristiques chroniques des élèves des fluctuations momentanées, révélant que si les relations sociales peuvent se réinitialiser partiellement entre deux années scolaires, les émotions négatives ont tendance à persister, voire à s'intensifier lors des transitions.

• Nécessité d'interventions multidimensionnelles : La simple prévention de l'intimidation est jugée insuffisante.

Les interventions doivent impérativement intégrer la promotion du bien-être et la gestion des émotions pour rompre les cycles de victimisation.

--------------------------------------------------------------------------------

1. État des lieux : Un portrait préoccupant chez les jeunes

Les données statistiques issues d'enquêtes canadiennes et québécoises révèlent une réalité complexe pour les élèves :

| Indicateur | Garçons | Filles | | --- | --- | --- | | Tristesse ou désespoir quotidien (début secondaire) | 19 % | 36 % | | Sentiment de ne pas être accepté tel que l'on est | 36 % | 52 % | | Victimes d'intimidation (12 derniers mois - Québec) | ~11 % | ~11 % |

Note sur la victimisation : Bien que le chiffre de 11 % soit cité, la proportion peut grimper jusqu'à 20 %, voire 40 % pour des événements isolés, soulignant la difficulté de cerner précisément ce phénomène.

--------------------------------------------------------------------------------

2. Définition des concepts fondamentaux

L'étude s'articule autour de trois réalités distinctes mais interconnectées :

• Émotions négatives : Comprennent la tristesse, le sentiment de désespoir et les idées négatives.

Elles sont considérées comme des précurseurs de la dépression, bien qu'elles ne correspondent pas nécessairement à un diagnostic clinique à ce stade (primaire).

• Isolement des pairs : Fait d'avoir peu d'interactions sociales, que ce soit par choix ou par rejet subi. Le rejet est la forme d'isolement non volontaire la plus fréquente.

• Victimisation : Actes d'agressivité intentionnels et répétitifs caractérisés par un déséquilibre des forces (physiques ou de réputation).

Elle peut être directe (frapper, insulter) ou indirecte (nuire à la réputation, propager des rumeurs).

--------------------------------------------------------------------------------

3. Modèles théoriques de la relation pairs-émotions

Trois modèles alternatifs tentent d'expliquer l'interaction entre ces variables :

1. Modèle des risques interpersonnels : Les expériences difficiles avec les pairs agissent comme des stresseurs qui s'accumulent et génèrent des émotions négatives.

C'est le modèle le plus testé et documenté à ce jour.

2. Modèle axé sur les symptômes : Les émotions négatives (ou l'affectivité négative) entraînent un retrait social ou une vulnérabilité qui fait de l'élève une cible privilégiée pour la victimisation.

3. Modèle transactionnel : Suppose une influence réciproque et un renforcement mutuel entre les émotions et les expériences sociales.

--------------------------------------------------------------------------------

4. Méthodologie de la recherche

L'étude a suivi 992 élèves de la 3e à la 6e année du primaire (Québec) sur deux années scolaires, avec quatre points de mesure.

L'originalité de l'approche réside dans l'utilisation de modèles statistiques ("modèles à décalage croisé avec intercept aléatoire") permettant de distinguer :

• Le Trait (stable/chronique) : La tendance d'un élève à être d'une certaine façon sur le long terme.

• L'État (changeant) : Les fluctuations d'un élève autour de sa propre tendance stable à un moment précis.

--------------------------------------------------------------------------------

5. Analyse des résultats : Des dynamiques différenciées

Interrelations stables (Traits)

De manière chronique, les trois dimensions sont liées : un élève ayant une tendance stable à l'isolement aura également une tendance stable à la victimisation et aux émotions négatives.

Ces réalités co-occurrent sans ordre temporel défini.

Dynamiques temporelles (États changeants)

L'analyse des fluctuations d'un moment à l'autre révèle des mécanismes distincts :

• Émotions négatives et Isolement : Suivent un modèle transactionnel.

Un niveau élevé d'émotions négatives en début d'année prédit un isolement accru en fin d'année, et inversement. C'est une boucle d'accentuation.

• Émotions négatives et Victimisation : Suivent un modèle axé sur les symptômes.

Les émotions négatives en début d'année prédisent une victimisation accrue plus tard, mais la victimisation ne semble pas augmenter les émotions négatives de manière immédiate.

Ce lien est direct et ne passe pas par l'intermédiaire de l'isolement.

• Stabilité temporelle :

◦ La victimisation et l'isolement sont plus stables au sein d'une même année qu'entre deux années.

Le changement de classe ou d'enseignant atténue l'effet de réputation.    ◦

Les émotions négatives sont plus stables entre les années scolaires, suggérant une anticipation anxieuse de la rentrée ou une persistance des traits internes malgré les changements d'environnement.

Constat important : Ces mécanismes sont identiques pour les garçons et les filles, ainsi que pour les élèves plus jeunes ou plus vieux au sein du primaire.

--------------------------------------------------------------------------------

6. Conclusions et orientations pour l'action

Pour la recherche

Les résultats de cette étude québécoise, bien que novateurs, ne font pas encore consensus au niveau international, d'autres études montrant parfois des résultats inverses ou sexués.

Une réplication du modèle est prévue en Belgique (Flandre) pour valider ces observations.

Pour l'intervention en milieu scolaire

L'étude remet en question les stratégies d'intervention uniquement centrées sur le comportement social :

• Insuffisance de la lutte contre l'intimidation seule : Retirer un élève d'une situation de victimisation ne garantit pas la disparition de ses émotions négatives.

• Approche multifactorielle : Il est impératif d'agir simultanément sur l'environnement social et sur le bien-être psychologique interne.

• Priorité à la promotion du bien-être : La prévention de la dépression et la gestion des émotions négatives dès le primaire sont des leviers essentiels pour réduire, par ricochet, les risques de victimisation et d'isolement.

"Les efforts de prévenir la victimisation sont essentiels, mais nos résultats suggèrent qu'ils ne sont potentiellement pas suffisants parce qu'il y a une dynamique plus large."

Version 4 of this preprint has been peer-reviewed and recommended by Peer Community in Genomics.<br /> See the peer reviews and the recommendation.

connecting earlier with now

!!!

LOVE

term

term

addressing it is taking that idea from arabiam nights

How could this experience help this person come closer to the Savior? What's an experience you've had with this that you could testify of?

If it seems like they have this down, skip and go to the next section!

What principle could help the person I'm talking to come closer to the Savior? What have you learned about the significance of that principle?

possible bias

does this relate to you? what is your screen time? what are you using your phone for? is it healthy or unhealthy?

Briefing : L’autorégulation chez les enfants victimes d’agression sexuelle

Résumé exécutif

Ce document synthétise les résultats de recherches doctorales portant sur l’autorégulation des enfants ayant survécu à une agression sexuelle (AS).

L’autorégulation, définie comme la capacité à moduler ses réponses cognitives et émotionnelles pour générer des comportements adaptatifs, est un processus clé souvent altéré par le trauma.

Les conclusions principales soulignent que si l’agression sexuelle est globalement associée à des difficultés de fonctionnement exécutif (inhibition et flexibilité cognitive), l'impact n'est pas uniforme.

La recherche identifie quatre profils distincts d'autorégulation chez les victimes : disrégulé, inhibé, flexible et régulation identifiée par les parents.

L'étude démontre également que des facteurs tels que le sexe de l'enfant, l'historique de maltraitance multiple et l'environnement socio-économique (défavorisation du quartier) influencent de manière significative les capacités d'autorégulation.

Les implications cliniques suggèrent d'abandonner les approches universelles au profit d'interventions différenciées et d'évaluations multi-méthodes (tâches cognitives et questionnaires) impliquant plusieurs répondants (parents et enseignants).

--------------------------------------------------------------------------------

1. Cadre théorique et définitions

L'agression sexuelle est une problématique de santé publique mondiale touchant environ une fille sur cinq et un garçon sur dix avant l'âge de 18 ans.

Elle entraîne des conséquences psychologiques variées, notamment des problèmes de comportement intériorisés (dépression, retrait) et extériorisés (agression, opposition).

L'autorégulation

Le concept d'autorégulation repose sur deux composantes interdépendantes :

• La régulation émotionnelle : Stratégies et compétences modulant l'expression et l'expérience des émotions.

• Les fonctions exécutives : Processus mentaux orientés vers un but, incluant :

◦ L'inhibition : Capacité à freiner une réponse automatique face à un stimulus (ex: répondre "nuit" quand on montre un soleil).    ◦ La flexibilité cognitive : Capacité à s'adapter au changement de règles dans l'environnement.

Le mécanisme biologique du trauma

L'exposition précoce à un stress intense (maltraitance, pauvreté) provoque une dysrégulation des hormones de stress, entraînant des atteintes structurelles et fonctionnelles au cerveau, ce qui fragilise les capacités d'autorégulation.

--------------------------------------------------------------------------------

2. Impact de l'agression sexuelle sur les fonctions exécutives

Les recherches présentées indiquent que l'agression sexuelle est un prédicteur significatif de difficultés exécutives, même après avoir contrôlé d'autres facteurs comme le TDAH ou la défavorisation sociale.

Constats par type de fonction

• Flexibilité cognitive : L'agression sexuelle est directement associée à une moins bonne performance dans les tâches mesurant cette capacité.

• Inhibition : Les enfants victimes montrent une performance significativement inférieure aux enfants non victimes.

Effet modérateur du sexe

L'étude révèle des différences marquées selon le sexe de l'enfant :

• Garçons : Les enseignants rapportent beaucoup plus de difficultés de fonctionnement exécutif chez les garçons victimes que chez les non-victimes. Ils affichent également des performances plus faibles aux tâches d'inhibition.

• Filles : Il y a peu de différence significative entre les filles victimes et non victimes sur le plan de l'évaluation des fonctions exécutives par les enseignants ou dans les tâches d'inhibition.

--------------------------------------------------------------------------------

3. Typologie des profils d'autorégulation

L'analyse a permis de dégager quatre profils types chez les enfants victimes d'agression sexuelle (échantillon de 225 enfants) :

| Profil | Proportion | Caractéristiques principales | Problèmes de comportement associés | | --- | --- | --- | --- | | Disrégulé | 39 % | Faible performance cognitive, forte labilité émotionnelle, difficultés rapportées par les parents. | Problèmes intériorisés et extériorisés élevés (comorbidité). | | Inhibé | 19 % | Excellente performance aux tâches d'inhibition, mais faibles compétences émotionnelles perçues par les parents. | Niveaux les plus élevés de problèmes intériorisés. | | Flexible | ~28 % | Autorégulation supérieure à la moyenne, profil concordant (maison/école), résilience. | Faible symptomatologie. | | Régulation (Parents) | 14 % | Performance cognitive faible, mais parents rapportant de très bonnes capacités (profil discordant). | Symptômes visibles par les enseignants mais sous-estimés par les parents. |

Analyse des profils spécifiques

• Le profil "Inhibé" : Ces enfants semblent utiliser une sur-régulation cognitive pour contrôler leurs impulsions, mais au prix d'une grande détresse interne.

Chez les filles, ce profil est un facteur de risque pour les problèmes intériorisés, tandis que chez les garçons, il semble agir comme un facteur de protection apparent contre les problèmes extériorisés.

• Le profil "Discordant" : Souvent associé à des agressions sexuelles intrafamiliales (80-90 % des cas dans ce groupe). Les parents peuvent surévaluer les compétences de l'enfant par désir de normalité ou sous l'effet d'un cadre familial trop rigide.

--------------------------------------------------------------------------------

4. Facteurs de risque et de protection contextuels

L'autorégulation ne dépend pas uniquement de l'acte traumatique, mais d'un écosystème de facteurs :

• Historique de maltraitance : Les profils "disrégulé" et "inhibé" sont corrélés à une exposition à un plus grand nombre de formes de maltraitance.

• Défavorisation du quartier : Les enfants vivant dans des quartiers favorisés présentent une meilleure autorégulation. Cela s'expliquerait par l'accès aux ressources (bibliothèques, musées, espaces verts) et une moindre exposition à la violence communautaire.

• Éducation parentale : Un niveau d'études plus élevé chez les parents favorise le développement des compétences langagières, lesquelles soutiennent directement l'autorégulation de l'enfant.

--------------------------------------------------------------------------------

5. Recommandations pour l'intervention clinique

Évaluation multidimensionnelle

Il est impératif de multiplier les sources d'information :

1. Multi-modalité : Combiner les questionnaires (perceptions) et les tâches cognitives (mesures objectives), car les résultats sont souvent divergents.

2. Multi-répondants : Inclure systématiquement le point de vue des enseignants pour identifier les difficultés qui pourraient être masquées dans le cadre familial.

Approche différenciée

L'intervention ne doit pas être identique pour tous les profils :

• Pour les enfants disregulés : Approche standard axée sur le renforcement des fonctions exécutives et de la régulation émotionnelle.

• Pour les enfants inhibés : Éviter de renforcer l'inhibition (potentiellement néfaste). Prioriser la reconnaissance, la compréhension et l'expression des émotions, ainsi que la flexibilité cognitive.

• Pour les enfants "flexibles" : L'intervention sur l'autorégulation peut être inutile. Se concentrer sur le soutien psychosocial et la prévention de la revictimisation.

• Pour le profil discordant : Évaluer la flexibilité des parents et utiliser des sources d'évaluation externes pour pallier la sous-estimation parentale des difficultés.

Pistes d'activités pratiques

• Pour l'inhibition : Jeux de type "1, 2, 3 Soleil", coloriage attentionnel (arrêter au signal), ou jeux de rôle où l'enfant doit attendre son tour face à une frustration.

• Pour la flexibilité : Jeux avec changement de règles fréquent (ex: varier qui gagne à "Roche-Papier-Ciseau"), résolution de problèmes avec des solutions multiples ou inversions de rôles.

• Implication des parents : Travailler sur l'autorégulation propre des parents et favoriser un attachement sécurisant, facteur de protection majeur pour l'enfant.

--------------------------------------------------------------------------------

Conclusion

La recherche souligne la complexité des trajectoires de développement après une agression sexuelle.

Le constat majeur est que le trauma n'entraîne pas systématiquement une dysrégulation.

Près de 42 % des enfants présentent des profils adaptés.

L'enjeu clinique réside dans l'identification des profils "surrégulés" ou "discordants", qui peuvent passer inaperçus tout en présentant des risques élevés de pathologie à long terme.

You need to learn to develop your writing skills regardless of your field of study or area of ​​interest. You'll most likely be asked to write a research paper during your academic career, so being able to write an essay is essential. Have a plan for how you'll gather information and implement it in your writing in a way that looks professional.

This is a really thoughtful way to approach it—focus on specific traits and needs, not labels. Different people can react very differently to the same app feature: autoplay videos, constant alerts, or crowded layouts might overwhelm one person but not another. Designing with flexibility (like mute options, reduced motion, clear layouts, and notification control) helps more people feel comfortable and included.

Being specific when writing is important, both for your readers and for yourself. Know the order in which you will write and be specific about the key points of the text.

Although modern scientists have a very scientific way to explain why so many people have abduction experiences with aliens, it is still worth exploring why it is aliens and not something else. Why do so many people have the same descriptions? Maybe later people are influenced by alien stories. How did the first people have the alien experience?

Preparation implies developing competency in the secure management of information, which makes it possible to establish levels of perfection and mastery in the protection of data.

Pourquoi les italiques et les majuscules ?

Comportements Parentaux Disrégulés et Fonctionnement des Enfants Victimes de Maltraitance : Document de Synthèse

Résumé Analytique

Ce document synthétise les résultats d'une thèse doctorale portant sur les liens entre les comportements parentaux disrégulés (CPD) et le développement socio-émotionnel de jeunes enfants suivis par les services de protection de la jeunesse.

L'analyse met en lumière un cycle de transmission intergénérationnelle de la maltraitance : les parents ayant vécu des traumatismes durant leur propre enfance sont plus susceptibles de manifester des comportements parentaux atypiques, effrayants ou intrusifs.

Les conclusions majeures de la recherche indiquent que :

1. Impact des CPD : Des niveaux élevés de comportements parentaux disrégulés sont directement associés à l'attachement désorganisé et à des problèmes de comportement (intériorisés et extériorisés) chez l'enfant.

2. Effet Protecteur : L'attachement sécurisant agit comme un modérateur crucial, protégeant l'enfant des impacts néfastes des CPD sur son développement comportemental.

3. Efficacité de l'Intervention : L'Intervention Relationnelle (IR), basée sur la rétroaction vidéo, réduit significativement la sévérité des comportements parentaux disrégulés, offrant ainsi une avenue clinique prometteuse pour les services de protection de l'enfance.

--------------------------------------------------------------------------------

1. Caractérisation des Comportements Parentaux Disrégulés (CPD)

Les comportements parentaux disrégulés sont des manifestations atypiques et perturbatrices qui surviennent lors des interactions avec l'enfant, particulièrement face à sa détresse.

Ces comportements sont souvent observés chez les parents signalés pour abus ou négligence.

Typologie des comportements selon l'échelle AMBIANCE

La recherche s'appuie sur la mesure AMBIANCE pour catégoriser cinq sous-types de comportements disrégulés :

| Sous-type de comportement | Description | | --- | --- | | Erreurs de communication affective | Minimiser, ignorer ou répondre de manière inappropriée à la détresse (ex: rire ou imiter l'enfant qui pleure). | | Confusion des rôles | Le parent aborde l'enfant comme s'il devait répondre aux propres besoins du parent (renversement de rôle) ou traite l'enfant comme un partenaire intime. | | Comportements effrayants ou apeurés | Manifestations d'effroi face aux besoins de l'enfant ou adoption d'une posture menaçante. | | Intrusion et négativité | Hostilité physique ou verbale, contrôle excessif des mouvements ou des interactions. | | Retrait | Création active d'une distance physique ou verbale, position d'impuissance et évitement de l'enfant lors des réunions. |

Le paradoxe de la peur sans solution

Ces comportements placent l'enfant dans un paradoxe insoluble.

La source habituelle de réconfort (le parent) devient simultanément la source de menace ou de détresse.

L'enfant ne peut donc pas élaborer de stratégie cohérente pour réguler son stress, ce qui mène à une désorganisation de l'attachement.

--------------------------------------------------------------------------------

2. Analyse des Impacts Développementaux et Facteurs de Protection

L'étude de 70 familles signalées au centre jeunesse de Montréal révèle les dynamiques entre l'exposition aux CPD et le fonctionnement de l'enfant.

Corrélations entre CPD et dysfonctionnement

L'exposition à des niveaux élevés de CPD est associée à :

• L'attachement désorganisé : Présent chez 50 % des enfants de l'échantillon.

• Problèmes de comportement : Augmentation des comportements agressifs (extériorisés) et des symptômes de retrait ou d'anxiété (intériorisés).

• Difficultés sociales et cognitives : Méfiance envers autrui, difficultés d'apprentissage et déficits de régulation émotionnelle.

L'attachement sécurisant comme bouclier

Un résultat central de la recherche montre que l'attachement sécurisant joue un rôle de facteur de protection.

• Pour les enfants ayant un attachement insécurisant, il existe un lien direct et significatif entre la sévérité des CPD et la présence de problèmes de comportement.

• À l'inverse, chez les enfants ayant un attachement sécurisant, ce lien n'est pas significatif.

Ces enfants présentent moins de problèmes de comportement malgré l'exposition aux mauvais traitements ou aux CPD.

--------------------------------------------------------------------------------

3. L'Intervention Relationnelle (IR) : Mécanismes et Efficacité

La recherche a évalué l'efficacité de l'Intervention Relationnelle par rapport aux services habituels (psycho-éducatifs).

Protocole de l'intervention

L'IR se déroule généralement sur 8 séances d'environ 1h30 et utilise la rétroaction vidéo comme levier de changement :

1. Discussion thématique : Aborde le rôle parental et le développement de l'enfant.

2. Période de jeu filmée (10-15 min) : Le parent réalise une activité spécifique avec une consigne orientée (ex: "observez votre enfant et décrivez ce qu'il fait").

3. Rétroaction vidéo : L'intervenant souligne les forces du parent et ses comportements sensibles.

Cela permet au parent de constater l'impact positif de ses actions sur son enfant (contacts visuels, rires, apaisement).

Résultats cliniques

L'intervention a démontré une réduction significative de plusieurs types de CPD comparativement au groupe contrôle :

• Diminution des erreurs de communication affective.

• Diminution des comportements d'intrusion.

• Diminution des comportements de retrait.

• Amélioration du score global de régulation parentale.

Note : Les comportements apeurés/effrayants et la confusion des rôles se sont révélés plus difficiles à modifier, étant plus subtils et moins facilement identifiables par le parent lors de la rétroaction vidéo.

--------------------------------------------------------------------------------

4. Implications pour les Services de Protection

L'étude conclut à la nécessité d'intégrer l'évaluation des CPD dans les pratiques cliniques courantes.

• Utilisation d'outils adaptés : L'adoption de l'instrument AMBIANCE brief est recommandée pour permettre aux intervenants de terrain de repérer les CPD sans nécessiter les protocoles lourds de recherche.

• Ciblage de l'attachement : Les interventions doivent viser prioritairement la sécurité d'attachement comme levier pour atténuer les conséquences des traumatismes.

• Formation continue : Former les intervenants à la reconnaissance des signaux de disrégulation subtils (hésitations, expressions faciales, postures) pour mieux accompagner les parents dans la réparation des interactions perturbées.

En résumé, l'Intervention Relationnelle s'avère être un outil puissant non seulement pour optimiser la sensibilité parentale, mais aussi pour réduire les placements à l'extérieur du milieu familial en améliorant la qualité fondamentale du lien parent-enfant.

I have noticed I feel like I have a more limited cognitive load space during lectures when my professor uses really active slides. I've gotten distracted or lost during instruction before, and felt like a "bad" student. As pretty and fun as they are, animations often distract me, and it can be hard to note-take when the audio doesn't match the transcriptions, as mentioned above. This felt validating!

As someone who color codes things often, I wouldn't have considered this on my own! I always thought lighter colors were better for highlighting because it allows me to see more text, but I can see how different students will have different visual/contrast needs. I never knew that bolding and underlining text were recommended over colored text or highlight tools.

>> https://hypothes.is/a/wmE7CAw6EfGOp-cQSnKXwA

<< https://hypothes.is/a/f_SKPAw5EfGFJSeD7U-tww

dweb.archive

From intelligence to autopoiesis: rethinking artificial intelligence through systems theory

xx

But the question is will trump leave behind the urban centers that screwed over reagan? does he even need them?

This is whats being studied in the other paper

Fickle, easily changed

At least in the industrial midwest, basically the republicnas help coause the decay but then the dems turn a blind eye to it which makes midwesterns ultimatly turn back towards rep